Biology of Reproduction

Published by Society for the Study of Reproduction
Online ISSN: 1529-7268
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Article
The purposes of this study were to determine the separate and interactive functions of progesterone and estradiol in regulating the cervical prostaglandin (PG) system in pregnant sheep at 0.7 gestations. At 106-108 days of gestational age (dGA), ewes were treated with vehicle for 14 days (n = 5) or vehicle for 12 days followed by estradiol 5 mg twice a day, intramuscularly for 2 days (n = 5) or progesterone 100 mg, twice a day, intramuscularly for 14 days (n = 5) or progesterone 100 mg twice a day, intramuscularly for 10 days and then 2 days vehicle followed by estradiol 5 mg twice a day intramuscularly for 2 days (n = 5). At 121-123 dGA, cervical tissues were obtained under halothane anesthesia. Cervical RNA and protein were extracted and analyzed for prostaglandin-endoperoxide synthase 2 (COX2), two PGE(2) receptors, PTGER2 and PTGER4, and estrogen receptor alpha (ESR1) by Northern and Western blot analysis. Immunocytochemistry and in situ hybridization were applied to localize cellular distribution of COX2, PTGER2, and PTGER4 in the cervix. Data were analyzed by ANOVA. COX2 and PTGER4 mRNAs and proteins were increased (P < 0.05) in ewes treated with combined estradiol and progesterone but not in ewes treated with estradiol or progesterone alone compared with controls. ESR1 mRNA was increased in ewes treated with progesterone and estradiol plus progesterone. In contrast, PTGER2 mRNA and protein remained the same after all treatments. COX2 mRNA and protein were localized only in cervical glandular epithelial cells, whereas PTGER2 and PTGER4 were localized in both cervical glandular epithelial and smooth muscle cells. In conclusion, these data suggest that additional progesterone priming at 0.7 gestations synergizes with estradiol to induce cervical COX2, PTGER4, and ESR1 and support our hypothesis that stimulation of the cervical PG system by estradiol is optimized by sufficient progesterone priming in the pregnant sheep cervix.
 
Article
In previous studies, we found that the human estrogen-regulated heat shock protein (hsp) 27 (human homologue of rat hsp25) is modulated in the endometrium during the different phases of the menstrual cycle and that it is present in endometrial predecidual cells and in decidual cells attached to the placenta. In the present report, we describe the cell type-specific pattern of hsp25 expression in the rat uterus during the periimplantation period as well as during early and late decidualization and placentation. The hsp25 expression pattern was also analyzed in pseudopregnant rats with deciduomas. Immunocytochemistry was performed with an antibody generated against a chimeric hybrid protein containing the N-terminal of the murine hsp25 and the C-terminal of the human hsp27. During pregnancy at the time of implantation, hsp25 was expressed in the endothelial cells of the endometrial vessels and in the luminal epithelium of the antimesometrial region. As pregnancy advanced, hsp25 appeared in predecidual/decidual cells close to the implantation region and then expanded to the mesometrial region. This expression pattern was very similar during pseudopregnancy. Hsp25 was strongly expressed in trophoblastic giant cells beginning on Day 11 of gestation; less expression was noted in the junctional and labyrinth zones of the chorioallantoic placenta (in some cells lining the vascular spaces). In all the disparate cell types that expressed hsp25, the presence of the protein did not correlate with cell proliferation or with apoptosis but with the state of differentiation. Some placental PRL-family members with molecular weights similar to that of hsp25 are also present in antimesometrial decidua and in differentiated trophoblast giant cells; therefore, in this study we eliminated the possibility that our antibody was recognizing prolactin. We also determined that the hybrid hsp25/27 protein did not bind prolactin receptors, and noted that the hsp25 immunostaining pattern was not identical to that of decidual prolactin. In conclusion, the striking cell type-specific timing of hsp25 expression points to hsp25 as a molecule that is important during the implantation, decidualization, and placentation processes.
 
Schematic drawing of the XbaI-EcoRV genomic DNA sequence of HLA-G. The HLA-G*0105N sequence corresponds to that of HLAG*010102, except for the deletion of cytosine, indicated by an arrow in exon 3 (C). The HLA-G gene consists of eight exons (E1-E8), corresponding, respectively, to the peptide leader domains (L), 1, 2, and 3, the transmembrane domain (TM), and the intracytoplasmic domain (cyt). Exons were separated by introns (i). The HLA-G*0105N gene putatively encodes the membrane-bound HLA-G2,-G3, and soluble HLA-G6 and-G7 proteins, in all of which exon 3, which contains the deletion, is deleted. HLA-G1,-G4, and-G5 might consist of the leader peptide, the complete 1 domain, the first half of the 2 domain, and a premature end in the following exon. 
DNA sequencing electropherogram of the HLA-G*010102 allele (A) and before deletion of the cytosine in exon 3 and after directed mutagenesis, yielding the HLA-G*0105N allele (B). 
Immunocytostaining of M8 transfectants with anti-HLA-G antibodies 4H84 (a-d), MEM-G/04 (e-h), MEM-G/09 (i-l), and 5A6G7 (m-p). All antibodies are negative in the control M8-pcDNA (a, e, i, m). The anti-HLA-G1,-G2,-G5,-G6 antibody MEM-G/04 is positive in all HLA-G transfected cells (f-h). The anti-HLA-G1,G5 antibody MEM-G/09 stained M8-HLAG*010102 (k) but not M8-HLA-G*0105N (j) or M8-HLA-G2 (l). The anti-HLA-G5,G6 antibody 5A6G7 stained M8-HLAG*0105N (n) and M8-HLA-G*010102 (o) but not M8-HLA-G2 (p). Original magnification 200. 
Detection by flow cytometry analysis of the HLA-G1 isoform on M8-HLA-G*010102 but not on M8-HLA-G*0105N. Cells were labeled by indirect immunofluorescence with MEM-G/09 mAb (bold profiles) and an isotope-matched control Ab (light profiles). M8-pcDNA and M8-HLA-G1 cells were used as controls. 
Article
Expression of the nonclassical HLA class I antigen, HLA-G, is associated with immune tolerance in view of its role in maintaining the fetus in utero, allowing tumor escape, and favoring graft acceptance. Expressed on invasive trophoblast cells, HLA-G molecules bind inhibitory receptors on maternal T lymphocytes and NK cells, thereby blocking their cytolytic activities and protecting the fetus from maternal immune system attack. The HLA-G gene consists of 15 alleles, including a null allele, HLA-G*0105N. HLA-G*0105N presents a single base deletion, preventing translation of both membrane-bound (HLA-G1) and full-length soluble isoforms (HLA-G5) as well as of the spliced HLA-G4 isoform. The identification of healthy subjects homozygous for this HLA-G null allele suggests that the HLA-G*0105N allele may generate other HLA-G isoforms, such as membrane-bound HLA-G2 and -G3 and the soluble HLA-G6 and -G7 proteins, which may substitute for HLA-G1 and -G5, thus assuming the immune tolerogeneic function of HLA-G. To investigate this point, we cloned genomic HLA-G*0105N DNA and transfected it into an HLA-class I-positive human cell line. The results obtained indicated that HLA-G proteins were indeed present in HLA-G*0105N-transfected cells and were able to protect against NK cell lysis. These findings emphasize the role of the other HLA-G isoforms as immune tolerogeneic molecules that may also contribute to maternal tolerance of the semiallogenic fetus as well as tumor escape and other types of allogeneic tissue acceptance.
 
Article
Definite causes for several pathologies of pregnancy remain unknown. In light of several recent studies, however, diminished or aberrant HLA-G expression may be associated with certain complication of pregnancy and be linked to HLA-G polymorphism. We analyzed DNA from 60 normal placentas (controls), 140 placentas from miscarriage, 36 placentas from preeclampsia, 76 placentas from fetal hypotrophy, and 34 placentas with hypoxia for variations in coding regions (allelic groups G*0101 to G*0107) and the 14-bp deletion/insertion into the 3'-untranslated region. No statistically significant differences were observed in the distribution of allelic group between pathological placentas and controls with the exception of G*0106 allele frequency in preeclamptic compared with control placentas (21.2% and 6.6%, respectively). A greater frequency of this allele also was observed in the two subgroups of miscarriage and hypoxia compared with that in controls. In addition, presence of the 14-bp sequence was prominent in preeclampsia compared with controls (60.8% vs. 35%, respectively), and homozygotes with deletion were not detected in the pathology. The results suggest that the G*0106 allele, which is coupled with the presence of the 14-bp sequence, contributes and/or is a relevant marker in some specific complications of pregnancy, especially preeclampsia.
 
Article
The compounds WHI-05 (5-bromo-6-methoxy-5, 6-dihydro-3'-azidothymidine-5'-[p-methoxyphenyl] methoxyalaninyl phosphate) and WHI-07 (5-bromo-6-methoxy-5, 6-dihydro-3'-azidothymidine-5'-[p-bromophenyl] methoxyalaninyl phosphate) are aryl phosphate derivatives of zidovudine (ZDV) with dual-function anti-human immunodeficiency virus and contraceptive activity. These drugs were rationally designed to bypass the thymidine kinase (TK) dependency of ZDV activation as well as to achieve spermicidal activity. We investigated the TK activity and intracellular metabolism of WHI-05 and WHI-07 in normal human vaginal and cervical epithelial cells as well as sperm. The time- and concentration-dependent intracellular formation of ZDV metabolites following addition of WHI-05 and WHI-07 to normal human vaginal, ectocervical, and endocervical epithelial cells as well as motile sperm was studied by analytical HPLC. Thymidine kinase activity in these cells was determined by the flow cytometric method based on intracellular phosphorylation of the fluorescent nucleoside, 5-amino-2-deoxyuridine-dansyl chloride and by the ability of cell-free extracts to convert [(3)H]thymidine to thymidine monophosphate in comparison to NALM-6, a pre-B leukemia cell line. TK activity of genital tract epithelial cells and sperm was found to be relatively low or lacking. Addition of WHI-05 and WHI-07 to vaginal and cervical epithelial cells resulted in their concentration- and time-dependent conversion to alaninyl ZDV monophosphate (Ala-ZDV-MP) and 5'-ZDV monophosphate as the major metabolites. Studies using motile human sperm also demonstrated the conversion of WHI-05 and WHI-07 to Ala-ZDV-MP. These results demonstrate that human female genital tract epithelial cells and sperm efficiently convert WHI-05 and WHI-07 to bioactive ZDV metabolites despite their TK deficiency.
 
Comparative cytotoxic activity of spermicidal aryl phosphate derivatives of bromo-methoxy ZDV versus N-9 against normal human ectocervical and endocervical epithelial cells. 
Article
WHI-07, a novel bromo-methoxy-substituted aryl phosphate derivative of zidovudine (ZDV), is a potent dual-function contraceptive agent. Although the bromo-methoxy functional groups in the thymine ring of its ZDV are very important for its sperm-immobilizing activity (SIA), the importance of the esterification of the phosphate group with an amino acid side chain and the identity of the para substituent in the aryl moiety remain unclear. In the present study, we have synthesized 23 new analogues of WHI-07 by replacing the alanine (Ala) side chain with different amino acids containing nonpolar side chains, namely tryptophan (Trp), proline (Pro), phenylalanine (Phe), leucine (Leu), methionine (Met), valine (Val), or glycine (Gly). The para substituents on the aryl moiety included bromo, chloro, fluoro, nitro, or methoxy groups. The SIA of each of the 23 WHI-07 analogues was evaluated by computer-assisted sperm analysis. The potential cytotoxicity of these compounds against normal human ectocervical and endocervical epithelial cells was evaluated using MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) cell viability assays. The replacement of the Ala side chain of WHI-07 with Val, Leu, or Phe led to a complete loss of SIA (EC(50) values > 500 microM), whereas replacement with Trp reduced the SIA by 4-fold. The presence of para substituents on the phenyl moiety led to significant alterations in SIA. The anti-human immunodeficiency virus (HIV) activity of Trp-containing WHI-07 analogues was also diminished. Our finding highlights the necessity of Ala side chain and the presence of electron-withdrawing para-bromo substituent on the phenyl moiety in addition to bromo-methoxy functionalization groups on the thymine ring in order for the phosphoramidate derivatives of ZDV to be effective dual-function spermicidal agents. Unlike the detergent-type microbicide, nonoxynol-9, which was cytotoxic to normal human ectocervical and endocervical epithelial cells (IC(50) values of 22 microM and 16 microM, respectively) at spermicidal concentrations (EC(50) = 81 microM), WHI-07 and its active analogues were selectively spermicidal without cytotoxicity against female genital tract epithelial cells. WHI-07 and its Trp analogues hold particular clinical promise for the development of novel, nondetergent-type prophylactic contraceptives for the prevention of heterosexual HIV/acquired immunodeficiency syndrome transmission.
 
Article
Postnatal development of Leydig cells involves transformation through three stages: progenitor, immature, and adult Leydig cells. The process of differentiation is accompanied by a progressive increase in the capacity of Leydig cells to produce testosterone (T). T promotes the male phenotype in the prepubertal period and maintains sexual function in adulthood; therefore, disruption of T biosynthesis in Leydig cells can adversely affect male fertility. The present study was designed to evaluate the ability of a xenoestrogen, methoxychlor (the methoxylated isomer of DDT [1,1, 1-trichloro-2,2-bis(p-chlorophenyl)ethane]), to alter Leydig cell steroidogenic function. Purified progenitor, immature, and adult Leydig cells were obtained from, respectively, 21-, 35-, and 90-day-old Sprague-Dawley rats treated with graded concentrations of the biologically active metabolite of methoxychlor, 2, 2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), and assessed for T production. HPTE caused a dose-dependent inhibition of basal and LH-stimulated T production by Leydig cells. Compared to the control value, reduced T production by progenitor and immature Leydig cells was apparent after 10 h of HPTE treatment in culture; the equivalent time for adult Leydig cells was 18 h. The reversibility of HPTE-induced inhibition was evaluated by incubating Leydig cells for 3, 6, 10, 14, or 18 h and measuring T production after allowing time for recovery. After treatment with HPTE for 3 h, T production by immature and adult Leydig cells for the 18-h posttreatment period was similar to the control value, but that of progenitor Leydig cells was significantly lower. The onset of HPTE action and the reversibility of its effect showed that Leydig cells are more sensitive to this compound during pubertal differentiation than in adulthood. T production was comparable when control and HPTE-treated immature Leydig cells were incubated with pregnenolone, progesterone, and androstenedione, but HPTE-treated Leydig cells produced significantly reduced amounts of T when incubations were conducted with 22R-hydroxycholesterol (P < 0.01). This finding suggested that HPTE-induced inhibition of T production is related to a decrease in the activity of cytochrome P450 cholesterol side-chain cleavage enzyme (P450(scc)) and cholesterol utilization. The reduced steady-state mRNA level for P450(scc) in HPTE-treated Leydig cells was demonstrated by reverse transcription-polymerase chain reaction and densitometry. In conclusion, this study showed that HPTE causes a direct inhibition of T biosynthesis by Leydig cells at all stages of development. This effect suggests that reduced T production could be a contributory factor in male infertility associated with methoxychlor and, possibly, other DDT-related compounds.
 
Article
We have recently reported that tetrahedral metallocene complexes containing vanadium(IV) (vanadocene) have potent spermicidal activity against human sperm. The spermicidal activity was dependent on vanadium(IV) as the central metal ion within the bis-cyclopentadienyl (Cp2)-metal complex, but the variation of diacido groups and/or replacement with bidentate ligands coordinated to the Cp2-vanadium(IV) moiety also significantly modulated the spermicidal potency. To assess the structure-activity relationship between vanadocenes and other coordination complexes of vanadium(IV), a set of 11 oxovanadium(IV) complexes with different geometrical configurations were synthesized and evaluated for spermicidal activity by computer-assisted sperm analysis. These complexes included mono and bis ancillary ligands, 1,10-phenanthroline (phen): [VO(phen), VO(phen)2, VO(Me2-phen), VO(Me2-phen)2, VO(Cl-phen), and VO(Cl-phen)2]; 2,2'-bipyridyl (bipy): [VO(bipy), VO(bipy)2, VO(Me2-bipy), and VO(Me2-bipy)2], linked via nitrogen atoms; and 5'-bromo-2'-hydroxyacetophenone (acph): [VO(Br,OH-acph)2], linked via oxygen donor atoms. All 11 oxovanadium(IV) complexes elicited concentration-dependent spermicidal activity at micromolar concentrations (EC50 values: 5.5-118 microM). The bis-phenanthroline complex of oxovanadium(IV), VO(Cl-phen)2, was the most active, and the mono bipyridyl complex, VO(bipy), was the least active; the order of efficacy was VO(Cl-phen)2 > VO(phen)2 > VO(Br,OH-acph)2 > VO(Me2-phen) > VO(bipy)2 > VO(phen) > VO(Cl-phen) > VO(Me2-phen)2 > VO(Me2-bipy)2 > VO(Me2-bipy) > VO(bipy). The neutral complex, VO(Br, OH-acph)2, induced rapid sperm immobilization (T1/2 = 38 sec). The sperm-immobilizing activity of mono- and bis-ligated oxovanadium(IV) complexes was irreversible, since the treated sperm underwent apoptosis, as determined by the flow cytometric quantitation of mitochondrial membrane potential, surface Annexin V binding assay, and in situ DNA nick-end labeling of sperm nuclei. The percentages of apoptotic sperm quantitated by the flow cytometric assay correlated well with the spermicidal potency of oxovanadium(IV) complexes. These results provide unprecedented evidence that the spermicidal and apoptosis-inducing activities of vanadium(IV) complexes are determined by the oxidation state of vanadium as well as their geometry. Because of its rapid and potent sperm-immobilizing activity, the bromo-hydroxyacetophenone complex, [VO(Br,OH-acph)2], may be useful as a contraceptive agent.
 
Article
Aneuploidy in germ cells results in reproductive failure and mental and physical disorders in humans. Unfortunately, little is known about the causes and mechanisms of aneuploidy induction. The objective of this study was to test the hypothesis that propylene glycol (1,2-propanediol; PG) induces cytogenetic aberrations in mouse metaphase II (MII) oocytes that predispose zygotes to aneuploidy. Female ICR mice received 7.5 IU eCG and 5.0 IU hCG 48 h later. PG doses of 1300, 2600, and 5200 mg/kg body weight were given 3 h post-hCG; controls received the solvent deionized water. Ovulated oocytes were collected 16 h after administration of PG and processed for cytogenetic analysis. For the one-cell zygote cytogenetic study, females were given PG and paired (1:1) with ICR males for 16 h. Females that mated were given 2 x 10(-3) M colchicine 22 h post-PG, and zygotes were collected 18 h later. PG significantly (p < 0.05) increased both the proportion of MII oocytes with premature centromere separation (PCS) and the proportion of aneuploid one-cell zygotes. These results support the hypothesis that PG-induced PCS in MII oocytes predisposes zygotes to aneuploidy.
 
Article
The pesticide 1,2-dibromo-3-chloropropane (DBCP) is a known male reproductive toxin. Previous studies employed production-grade DBCP containing allyl chloride and epichlorohydrin, both capable of producing effects similar to DBCP. The purpose of this study was to determine if purified DBCP caused the same effects as DBCP containing allyl chloride. Rats were injected for 6 mo with varying doses of pure or production-grade DBCP. Very few differences were observed in the parameters measured between pure and production-grade DBCP-treated animals at any one dose level. Treatment with 25 mg/kg DBCP, pure or production grade, reduced body weight as well as the weight of the testes, prostate glands and seminal vesicles, and elevated serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH). This dose of pure DBCP reduced serum testosterone. Treatment with 5 mg/kg of either grade DBCP caused decreases only in body and testis weights. Animals treated with 1 mg/kg did not show any major differences from controls. These results indicate that DBCP, and probably not a contaminant, is responsible for the effects observed on male reproduction. Furthermore, DBCP appears to affect either androgen action or production since multiple androgenic indices are affected by DBCP administration.
 
Article
Macrophages accumulate within stromal tissue subjacent to the luminal epithelium in the mouse uterus during early pregnancy after seminal fluid exposure at coitus. To investigate their role in regulating epithelial cell expression of fucosylated structures required for embryo attachment and implantation, fucosyltransferase enzymes Fut1, Fut2 (Enzyme Commission number [EC] 2.4.1.69), and Fut4 (EC 2.4.1.214) and Muc1 and Muc4 mRNAs were quantified by quantitative real-time PCR in uterine epithelial cells after laser capture microdissection in situ or after epithelial cell coculture with macrophages or macrophage-secreted factors. When uterine macrophage recruitment was impaired by mating with seminal plasma-deficient males, epithelial cell Fut2 expression on Day 3.5 postcoitus (pc) was reduced compared to intact-mated controls. Epithelial cell Fut2 was upregulated in vitro by coculture with macrophages or macrophage-conditioned medium (MCM). Macrophage-derived cytokines LIF, IL1B, and IL12 replicated the effect of MCM on Fut2 mRNA expression, and MCM-stimulated expression was inhibited by anti-LIF and anti-IL1B neutralizing antibodies. The effects of acute macrophage depletion on fucosylated structures detected with lectins Ulex europaeus 1 (UEA-1) and Lotus tetragonolobus purpureas (LTP), or LewisX immunoreactivity, were quantified in vivo in Cd11b-dtr transgenic mice. Depletion of macrophages caused a 30% reduction in luminal epithelial UEA-1 staining and a 67% reduction in LewisX staining in uterine tissues of mice hormonally treated to mimic early pregnancy. Together, these data demonstrate that uterine epithelial Fut2 mRNA expression and terminal fucosylation of embryo attachment ligands is regulated in preparation for implantation by factors including LIF and IL1B secreted from macrophages recruited during the inflammatory response to insemination.
 
Article
Ovine placental tissue has been incubated with [4-¹⁴C]androstenedione and [1,2-³H2]-epitestosterone for 10, 30, 60, and 120 min. The subsequent radioactive content of the substrates and metabolites has been evaluated after each period of incubation and plotted against time. The rate of interconversion for the two substrates was examined as well as the formation of estrone, and the two epimers of estradiol. The incorporation of both labels into water soluble material was noteworthy whilst the formation of estrone sulfate from both substrates was considered of particular interest.
 
Article
Germ cell apoptosis, which occurs normally during spermatogenesis, increases after testosterone withdrawal from the testis. The molecular mechanism by which this occurs remains uncertain. The Fas system has been implicated as a possible key regulator of apoptosis in various cells: binding of Fas ligand (FasL), a type II transmembrane protein, to Fas, a type I transmembrane receptor protein, triggers apoptosis in cells expressing Fas. Recently, Fas has been localized to germ cells, and FasL to Sertoli cells, within the rat testis. We hypothesized that Fas protein content would rise in response to reduced levels of testosterone as part of a suicide pathway that would result in germ cell apoptosis. To test this hypothesis, ethane 1,2-dimethanesulfonate (EDS), a Leydig cell toxicant, was used to kill Leydig cells and thus reduce intratesticular testosterone levels in Sprague Dawley rats. Apoptosis was examined in situ and biochemically, and Fas protein content in the testis was monitored by Western blot analysis. We show that EDS injection results in the following sequence of events: apoptotic death of Leydig cells by a mechanism that does not involve Fas; reduced testosterone; increased testicular Fas content; and germ cell apoptosis. These results suggest that Fas may play a role in the apoptotic death of germ cells that results from reduced intratesticular testosterone levels, and that testosterone may play a role in germ cell survival via its suppression of Fas.
 
Article
The polycyclic aromatic hydrocarbon (PAH) 9,10-dimethyl-1,2-benzanthracene (DMBA) destroys primordial, primary, and secondary ovarian follicles in rodents, but its effects on antral follicles have received limited attention. PAHs are metabolized to reactive species, some of which can undergo redox cycling to generate reactive oxygen species (ROS). We previously showed that ROS initiate apoptosis in preovulatory follicles cultured without gonadotropin support and that glutathione (GSH) depletion induces apoptosis in the presence of gonadotropins. In the present study, we tested the hypothesis that DMBA induces apoptosis in preovulatory follicles, which is mediated by ROS and prevented by GSH. Preovulatory follicles were isolated from ovaries of 25-day-old rats 48 h after the injection of 10 IU of eCG and were cultured with DMBA in the presence of FSH for 2 to 48 h. DMBA induced granulosa cell (GC) and theca cell (TC) apoptosis at 48 h, as judged by TUNEL and activated caspase-3 immunostaining. DMBA treatment also increased the numbers of GCs and TCs that immunostained for the proapoptotic protein BAX. Follicular ROS levels were significantly increased in DMBA-treated follicles at 12, 24, and 48 h. GSH supplementation protected against and GSH depletion enhanced the induction of apoptosis in GCs and TCs by DMBA. These findings suggest that GSH is a critical protective mechanism against DMBA-induced apoptosis in antral follicles and that ROS generation may mediate DMBA-induced GC apoptosis.
 
Design of targeting constructs, and PCR and Southern blot screening strategies. The 5 end of an endogenous 1,3-GT allele (exons, solid boxes; introns, thick line) showing the regions of homology used for the 5 and 3 arms of the targeting constructs, the position of the probe (closed bar), and the HindIII and EcoRI restriction enzyme sites used for Southern blot analyses. Targeting constructs pGTIFneo and pGTKneo are shown with the promoterless neo poly(A) (open box) cloned in frame with the endogenous ATG or with a Kozak consensus sequence (open arrow), respectively. A targeted allele shows the position of primers (solid arrowheads) used for PCR analyses and the unique AscI site; * indicates in-frame or Kozak consensus sequence. Primers 7 and 8 amplified 3.4-kb and 2.7-kb targeted and endogenous bands, respectively. Primers 1 and 9 amplified 12.8-kb and 12.1-kb targeted and endogenous bands, respectively.
Summary of nuclear transfer results for the generation of 1,3- GT knockout male pigs. Donor cell lines C173 HT F210 HT F27 HT F29 HT
Flow cytometric analysis of pig fibroblasts. The x-axis shows the fluorescence intensity, and the y-axis shows the relative cell count for unstained cells, H and Gal1,3-Gal antigen expression (arrowheads). A) Expression profile for the parental fibroblast cell line C2.1 HT from which piglets 136- 1 HT , 136-3 HT , and 136-4 HT were derived. B) Expression profile for the parental fibroblast cell line F1.1 HT from which piglets 141-1 HT and 141-2 HT were derived. C) Expression profile for piglet 136-1 HT , which was positive for Gal1,3-Gal but not H antigen. D) Expression profile for piglet 141- 1 HT , which was positive for Gal1,3-Gal and H antigens.  
Nucelar transfer live piglets. A) Litter 136 produced by nuclear transfer using C173 HT and a nontargeted donor cell line: piglets 136-1 (1,3- GT knockout, white), 136-2 (1,3-GT WT, black), and 136-4 (1,3-GT knockout, white). Inset shows the entire litter at birth: three 1,3-GT knockout (white) and one WT (black) piglets. B) Two 1,3-GT knockout piglets from litter 141 cloned by nuclear transfer from cell line NF29 HT : piglets 141- 1 HT and 141-2 HT .  
Article
The production of genetically engineered pigs as xenotransplant donors aims to solve the severe shortage of organs for transplantation in humans. The first barrier to successful xenotransplantation is hyperacute rejection (HAR). HAR is a rapid and massive humoral immune response directed against the pig carbohydrate Galalpha 1,3-Gal epitope, which is synthesized by alpha 1,3-galactosyltransferase (alpha1,3-GT). The Galalpha 1,3-Gal antigen also contributes to subsequent acute vascular rejection events. Genetic modifications of donor pigs transgenic for human complement regulatory proteins or different glycosyltransferases to downregulate Galalpha 1,3-Gal expression have been shown to significantly delay xenograft rejection. However, the complete removal of the Galalpha 1,3-Gal antigen is the most attractive option. In this study, the 5' end of the alpha 1,3-GT gene was efficiently targeted with a nonisogenic DNA construct containing predominantly intron sequences and a Kozak translation initiation site to initiate translation of the neomycin resistance reporter gene. We developed two novel polymerase chain reaction screening methods to detect and confirm the targeted G418-resistant clones. This is the first study to use Southern blot analysis to demonstrate the disruption of the alpha 1,3-GT gene in somatic HT-transgenic pig cells before they were used for nuclear transfer. Transgenic male pigs were produced that possess an alpha 1,3-GT knockout allele and express a randomly inserted human alpha 1,2-fucosylosyltransferase (HT) transgene. The generation of homozygous alpha 1,3-GT knockout pigs with the HT-transgenic background is underway and will be unique. This approach intends to combine the alpha 1,3-GT knockout genotype with a ubiquitously expressed fucosyltransferase transgene producing the universally tolerated H antigen. This approach may prove to be more effective than the null phenotype alone in overcoming HAR and delayed xenograft rejection.
 
Article
Several drugs have been shown to cross the placental barrier and affect the fetal testis causing a reduction in testosterone with a resultant impairment of sexual differentiation and an ultimate problem in adult sexual function. In this study, pregnant female rats were treated with 25 mg/kg of the pesticide 1,2-dibromo-3-chloropropane (DBCP). Treatment began on Days 14.5, 16.5, or 18.5 and continued through Day 19.5 of gestation. Some animals were killed on Day 20.5 of intrauterine life and fetal intratesticular testosterone was measured. All other animals were allowed to deliver, and the males were raised to adulthood. At adulthood, body, testis, prostate glands and seminal vesicle weights were recorded. Intratesticular testosterone and luteinizing hormone (LH) receptors were measured. Male and female sexual behavior was quantified and the volume of the sexually dimorphic nucleus of the preoptic area of the hypothalamus was calculated. The histological appearance of the testis was also examined. Treatment for 6 days during fetal life with DBCP decreased intratesticular testosterone by 50% compared to controls at 20.5 days of gestation. At adulthood, all male rats treated during fetal life had a reduced body weight that was correlated with the duration of exposure. Adult testis weight was reduced to 75% of controls as a result of 2 days of fetal exposure to DBCP, whereas 4 and 6 days of exposure during fetal life reduced testis weight by greater than 90%. LH receptors and intratesticular testosterone, in the adults treated during fetal life, were also dramatically reduced.(ABSTRACT TRUNCATED AT 250 WORDS)
 
Number and characteristic of blastomeres in blastocysts produced in vitro from grade I immature oocytes exposed to various CPA conditions (propanediol, PrOH; ethanediol, EG) at the germinal vesicle stage. (Mean SD).
Developmental competence (7 days of culture in vitro) of in vitro-matured grade I oocytes after different cryoprotectant exposures (Propanediol, PrOH; ethanediol, EG) at the germinal vesicle stage. A) Percentage of cleaved embryos (solid bars) and blastocyst yield (number of blastocyst relative to total number of oocytes; open bars). B) Proportion of morula (solid bars) and blastocyst (open bars) stages relative to the total number of cleaved embryos (four replicates, values are mean SD; numbers above bars represent total number of oocytes per treatment). Among CPA exposure, bars with different letters differ (P 0.05).  
Article
This study assessed the impact of various cryoprotectant (CPA) exposures on nuclear and cytoplasmic maturation in the immature cat oocyte as a prerequisite to formulating a successful cryopreservation protocol. In experiment 1, immature oocytes were exposed to 0, 0.75, 1.5, or 3.0 M of 1,2-propanediol (PrOH) or 1,2-ethanediol (EG) at room temperature (25 degrees C) or 0 degrees C for 30 min. After CPA removal and in vitro maturation, percentage of oocytes reaching metaphase II (MII) was reduced after exposure to 3.0 M PrOH at 0 degrees C or 3.0 M EG at both temperatures. All CPA exposures increased MII spindle abnormalities compared to control, except 1.5 M PrOH at 25 degrees C. In experiments 2 and 3, immature oocytes were exposed to CPA conditions yielding optimal nuclear maturation that either had caused spindle damage (0.75 M PrOH, 1.5 M EG, and 3.0 M PrOH at 25 degrees C) or not (1.5 M PrOH at 25 degrees C). After maturation and insemination in vitro, oocytes were cultured for 7 days to assess treatment influence on developmental competence. CPA exposure did not affect fertilization, but the high incidence of MII spindle abnormalities resulted in a low percentage of cleaved embryos. Blastocyst formation and quality were influenced by both CPA types (EG was more detrimental than PrOH) and concentration (3.0 M was more detrimental than 1.5 M). Overall, cat oocytes appear to be highly sensitive to CPA except after exposure to 1.5 M PrOH at 25 degrees C, a treatment that still allowed approximately 60% of the oocytes to reach MII and approximately 20% to form blastocysts.
 
Article
Uterine leiomyomas (fibroids) are the most common benign tumors in women of reproductive age. These tumors are three to four times more prevalent in African American women, who also have a 10 times higher incidence of hypovitaminosis D than white women. Recent studies have demonstrated the antitumor effects of 1,25-dihydroxyvitamin D3 on several cancers, but its effects on uterine leiomyomas are still unknown. To determine the antitumor and therapeutic effects of 1,25-dihydroxyvitamin D3 on uterine leiomyomas, female Eker rats (14-16 mo old) harboring uterine leiomyomas were randomized into control and experimental groups and were given vehicle versus 1,25-dihydroxyvitamin D3 (0.5 μg/kg per day) subcutaneously for 3 wk, respectively. At the end of the experiment, the rats were euthanized, and the leiomyoma tumors were analyzed. Treatment with 1,25-dihydroxyvitamin D3 significantly reduced leiomyoma tumor size in Eker rats. It also reduced leiomyoma size by suppressing cell growth and proliferation-related genes (Pcna, cyclin D1 [Ccnd1], Myc, Cdk1, Cdk2, and Cdk4), antiapoptotic genes (Bcl2 and Bcl2l1 [Bcl-x]), and estrogen and progesterone receptors. Additionally, immunohistochemistry revealed decreased expression of PCNA and MKI67 (a marker of proliferation) and increased expression of caspase 3 in 1,25-dihydroxyvitamin D3-treated Eker rat leiomyomas. Toxicity analyses using serum samples showed similar levels of SGOT, SGPT, calcium, and total bilirubin in 1,25-dihydroxyvitamin D3-treated and vehicle-treated control Eker rats. These results support that 1,25-dihydroxyvitamin D3 is an antitumor agent that may be a potential safe, nonsurgical therapeutic option for the treatment of uterine leiomyomas.
 
Article
Uterine fibroids (leiomyoma) are the most common benign tumors associated with excessive deposition of extracellular matrix (ECM) that increase fibroid tumorigenicity. Herein, we determine the expression levels of vitamin D receptor (VDR) protein in human uterine fibroids and compared to adjacent normal myometrium. Using western blot analyses we found that at least 60% of uterine fibroids analyzed (25 out of 40) expressed low levels of VDR. The biologically active 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] function via binding to its nuclear VDR. We also found that 1,25(OH)2D3 induced VDR in a concentration-dependent manner, and reduced ECM-associated fibrotic and proteoglycans expressions in immortalized human uterine fibroid cell line (HuLM). At 1-10 nM concentrations, 1,25(OH)2D3 significantly induced (P<0.05) nuclear VDR, which was further stimulated by higher concentrations of 1,25(OH)2D3 in HuLM cells. 1,25(OH)2D3 at 10 nM concentration also significantly reduced (P<0.05) the protein expression of ECM-associated collagen type 1, fibronectin and plasminogen activator inhibitor (PAI-1) in HuLM cells. We also found that 1,25(OH)2D3 reduced mRNA and protein expressions of proteoglycans such as fibromodulin, biglycan and versican in HuLM cells. Moreover, the aberrant expression of structural smooth muscle actin fibers was reduced by 1,25(OH)2D3 treatment in a concentration-dependent manner in HuLM cells. Together, our results suggest that human uterine fibroids express reduced levels of VDR than the adjacent normal myometrium, and treatment with 1,25(OH)2D3 can potentially reduce the aberrant expression of major ECM-associated proteins in HuLM cells. Thus 1,25(OH)2D3 might be an effective, safe non-surgical treatment option for human uterine fibroids.
 
Article
The active form of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25[OH](2)D(3)) is a potent immunomodulatory seco-steroid. We have demonstrated that several components of vitamin D metabolism and signaling are strongly expressed in human uterine decidua from first trimester pregnancies, suggesting that locally produced 1,25(OH)(2)D(3) may exert immunosuppressive effects during early stages of gestation. To investigate this further, we used primary cultures of human decidual cells from first and third trimester pregnancies to demonstrate expression and activity of the enzyme that catalyzes synthesis of 1,25(OH)(2)D(3), 1alpha-hydroxylase (CYP27B1). Synthesis of 1,25(OH)(2)D(3) was higher in first trimester decidual cells (41 +/- 11.8 fmoles/h/mg protein) than in third trimester cells (8 +/- 4.4 fmoles/h/mg protein; P < 0.05). Purification of decidual cells followed by quantitative RT-PCR analysis showed that CYP27B1 was expressed by both CD10(+VE) stromal-enriched and CD10(-VE) stromal-depleted cells, with higher levels of mRNA in first trimester pregnancies. Expression of CYP27B1 correlated with TLR4 and IDO. Functional responses to 1,25(OH)(2)D(3) were studied using CD56(+VE) natural killer (NK) cells isolated from first trimester decidua. Decidual NK cells treated with 1,25(OH)(2)D(3) or precursor 25-hydroxyvitamin D(3) (25OHD(3)) for 28 h showed decreased synthesis of cytokines, such as granulocyte-macrophage colony stimulating factor 2 (CSF2), tumor necrosis factor, and interleukin 6, but increased expression of mRNA for the antimicrobial peptide cathelicidin antimicrobial peptide. These data indicate that human decidual cells are able to synthesize active 1,25(OH)(2)D(3), particularly in early gestation, and this may act in an autocrine/paracrine fashion to regulate both acquired and innate immune responses at the fetal-maternal interface.
 
Article
Theophylline (THP) and 1,3-dinitrobenzene (DNB) are thought to induce infertility by incapacitating the nurturing Sertoli cells and causing germ cell apoptosis in the testicular seminiferous epithelium, respectively. We hypothesized that THP and DNB exposure would alter the expression of the genes within the ubiquitin-proteasome pathway (UPP), implicated in spermatogenesis and epididymal sperm quality control. Rats were fed 0 or 8000 ppm of THP and necropsied on Days 18, 30, and 42 or administered 0, 2, or 6 mg/kg DNB via oral gavage and necropsied on Day 7. Tissues were collected from the testis and the caput, corpus, and cauda regions of the epididymis for transcriptional profiling by semiquantitative RT-PCR, real-time RT-PCR, and histopathology. Target UPP genes included those encoding for constitutive the 20S proteasomal core subunits Psmb1 (beta1), Psmb2 (beta2), and Psmb5 (beta5); the inducible 20S core subunits Psmb9 (LMP2), Psmb8 (LMP7), and Psmb10 (LMP10); and Ube1 (ubiquitin-activating enzyme E1), Ube2d3 (ubiquitin-conjugating enzyme E2), and Uchl1 (ubiquitin C-terminal hydrolase PGP9.5). Spermatozoa were collected from the cauda epididymis for analysis by light microscopy and flow cytometric evaluation of sperm surface ubiquitin. These data show that reprotoxic exposure alters the tissue-specific expression of UPP genes in the testis and epididymis, which may contribute to the aberrant spermatogenesis and epididymal processing of both normal and defective spermatozoa. Transcriptional profiling and flow cytometric analysis of the UPP thus captures the prodromal effects of reproductive toxicity not captured by conventional histology and functional cytology. Complementing seminal analysis with these measures may be useful in screening drug-induced toxicity or environmental infertility.
 
Agarose gel electrophoretic pattern of the CK isoforms. A) Muscle extract. B) Sperm extract of infertile men. C) Sperm extract of fertile men. Sperm CK isoforms do not form the MB heterodimer 1201. 
Article
Sperm creatine phosphokinase (CK) concentrations and the synthesis of the CK-M isoform reflect normal spermiogenesis and predict maturity and fertilizing potential of ejaculated human spermatozoa. Immature spermatozoa, characterized by cytoplasmic retention and low CK-M to CK-B isoform ratios, are deficient in zona binding and fail to cause pregnancies. Because these sperm lack zona-binding ability, we examined in this study whether beta 1,4-galactosyltransferase (GalTase), a key element of sperm-zona interactions in mice, is diminished in immature human sperm. Unexpectedly, GalTase was overexpressed in immature sperm relative to mature sperm: the levels of cytoplasmic CK and plasma membrane GalTase were positively correlated (r = 0.78, p < 0.001, n = 88). Sperm populations with various levels of cellular maturity, prepared by Percoll gradients, had different CK and GalTase concentrations, but within each subpopulation the relationship between CK and GalTase was maintained (p < 0.01-0.001). GalTase activities in intact and vortex-disrupted sperm fractions were similar, showing that GalTase is present on the surface membrane of human sperm--similar to the situation in all other species assayed. The changes previously reported by our laboratory in zona-binding ability and lipid peroxidation rates (which occur simultaneously with cytoplasmic extrusion), decline in CK activity, and increased expression of the CK-M isoform are suggestive of a remodeling of the sperm surface concomitant with cytoplasmic maturation. The changes reported here in GalTase expression on the surface of maturing spermatozoa prove this hypothesis.
 
Activation of control and IP 3 R-1 DR mammalian eggs injected with pSE. 
Characteristics of IP 3 -induced Ca 2 release in IP 3 R-1 DR bovine eggs. 
The threshold for IP 3-induced Ca 2 release is decreased in IP 3 R-1 DR bovine eggs. Control (left) and IP 3 R-1 DR (right) eggs were injected with IP 3 at a pipette concentration of 500 M (A and B, n 7 and 8, respectively), or 20 M (C and D, n 8 and 8, respectively) at 2024 hpm. The amplitude of the [Ca 2 ] i rise in IP 3 R-1 DR eggs is affected at a lower concentration of IP 3 (20 M, D), but not at saturating concentrations (500 M, B). Arrows indicate time of injection. 
Article
It is widely believed that stimulation of the phosphoinositide pathway and production of 1,4,5-inositol trisphosphate (IP(3)) underlies the oscillatory changes in the concentration of intracellular free calcium ions ([Ca(2+)](i)) seen during mammalian fertilization. IP(3) promotes Ca(2+) release in eggs by binding to its receptor, the type-1 IP(3) receptor (IP(3)R-1, also known as ITPR1), a ligand-gated Ca(2+) channel located in the membrane of the endoplasmic reticulum, the main Ca(2+) store of the cell. While IP(3)R-1 has been shown to mediate all Ca(2+) release during mouse fertilization, whether or not it plays such an essential role in fertilization-induced Ca(2+) release in large domestic species such as bovine and porcine is presently not known. Accordingly, we have generated metaphase II bovine eggs with a approximately 70%-80% reduction in the number of intact IP(3)R-1 by inducing receptor down-regulation during oocyte maturation. We did so by injecting the nonhydrolyzable IP(3) analogue, adenophostin A. Functional Ca(2+) release analysis revealed that IP(3)R-1 is the predominant Ca(2+) release channel in bovine eggs, requiring as little as 20% of total intact receptor to mount persistent [Ca(2+)](i) oscillations in response to fertilization, expression of PLCzeta (also known as PLCZ1), and adenophostin A. However, lower concentrations of IP(3) and near-physiological concentrations of porcine sperm extract were unable to trigger [Ca(2+)](i) oscillations in this reduced IP(3)R-1 model. Furthermore, we present evidence that the sensitivity of bovine IP(3)R-1 is impaired at the first embryonic interphase. Together, these results demonstrate the essential role of IP(3)R-1-mediated Ca(2+) release during fertilization in bovine eggs, and identify cell cycle regulatory mechanisms of [Ca(2+)](i) oscillations at the level of IP(3)R-1.
 
Article
Smooth muscle-mediated expansion and contraction of the vascular sinusoids of the corpora cavernosa may modulate male erectile function. To elucidate the biochemical events that control erection by promoting or inhibiting contraction of cavernosal smooth muscle, tissue from a potent man was grown in cell culture. The cells grew as noncontractile cultures, but had the following smooth muscle cell properties: These cells expressed desmin, the muscle cell-specific intermediate filament protein. They accumulated 45Ca2+ from the medium, which was released by exposure to the ionophore A23187, to cyclic nucleotides (cyclic guanosine 5'-monophosphate [GMP] much greater than cyclic adenosine 3',5'-monophosphate [AMP]), and to the phosphodiesterase inhibitor, papaverine; and; they accumulated Ca2+ in an ATP-dependent manner when the cultured cells were permeabilized by digitonin extraction. ATP-dependent Ca2+ uptake was inhibited approximately 80% by ruthenium red and simulated by cyclic GMP much greater than cyclic AMP. Inositol 1,4,5-trisphosphate (IP3), which is thought to mediate the release of Ca2+ by the smooth muscle cell sarcoplasmic reticulum in vivo, released approximately 0.85 pmol Ca2+/million cells from the digitonin-extracted cells. IP3-dependent release occurred in the presence of ruthenium red and was not affected by cyclic GMP or cyclic AMP. These results indicate that smooth muscle from this human source can be grown successfully in cell culture and that the biochemical pathways that regulate tension in vivo may be perpetuated in vitro. Moreover, some of the clinical responses to drugs administered in situ for erectile dysfunction (e.g. papaverine) may be the result of altered cavernosal smooth muscle cell Ca2+ exchange and may be mediated by cyclic GMP.
 
Article
The regulation of the phospholipase C (PLC) and the expression of inositol 1,4,5-trisphosphate receptors (IP(3)Rs) in terms of mRNA, proteins, and binding capacity were examined in the rat myometrium and endometrium at midgestation (Day 12) and at term (Day 21) comparatively to the estrogen-treated tissues (Day 0). In both uterine tissues, the production of inositol phosphates mediated by carbachol as well as by AlF(4)(-) was enhanced with advancing gestation. (3)[H]IP(3) binding sites in membranes also increased during pregnancy (Day 21 > Day 12 > Day 0). The mRNAs encoding for three isoforms of IP(3)R as well as their corresponding proteins, IP(3)R-1, IP(3)R-2, and IP(3)R-3 were coexpressed, albeit to different extents, in the myometrium and endometrium. The expression of IP(3)Rs increased with advancing gestation, except for IP(3)R-2 that increased only in the endometrium at term. Thus, the pregnancy-related upregulation of the PLC cascade coincided with an increase in the expression of IP(3)Rs. The difference noted between the two uterine tissues suggests that IP(3)Rs may have cell-specific functions.
 
Time-dependent changes in the percentage of eggs undergoing anaphase. Anaphase was determined by in situ DNA/chromatin staining at 13, 16, and 22 h post-hCG administration. Anaphase was not detected in any eggs at 13 h. Each time point represents at least 3 egg collections on different days. (In two of these collections, females of the same age were used and eggs were collected at the three indicated times after hCG, resulting in data essentially the same as those presented above.) Total number of eggs: 13 h, 112; 16 h, 209; 22 h, 123. 
Time-dependent increase in the ZP2 conversion. Eggs were retrieved at the indicated times following hCG administration, and the ZP2to-ZP2, conversion was determined as described in Materials and Methods. The experiment was performed three times, and 40-44 ZP were analyzed in each group. The data are expressed as the mean ± SEM. 
Article
With increasing time after ovulation, mammalian eggs become more sensitive to agonists of activation in vitro or may undergo spontaneous activation in vivo. We have tested the hypothesis that postovulatory eggs undergo time-dependent cell cycle and cytoplasmic changes that result in a partially activated state, accounting for their time-dependent susceptibility to activate. In vivo changes in key activation markers in mouse eggs were quantified at 13, 16, and 22 h post-hCG (1, 4, and 10 h postovulation). Spontaneous activation was first detected at 16 h, with a 20-25% decrease in the activities of histone H1 and mitogen-activated protein (MAP) kinases and with 3% of eggs undergoing both anaphase onset and a partial loss of cortical granules. By 22 h, more than 60% of eggs were in anaphase, H1 and MAP kinase activities had decreased 40-50%, the extent of zona pellucida modification had increased, and proteins normally synthesized after fertilization had appeared. Pronuclear formation in response to inositol 1,4,5-trisphosphate injection increased dramatically from 10% at 13 h to about 40% and 90% at 16 h and 22 h, respectively. The partial decreases (less than those after fertilization) in H1 and MAP kinase activities provide a likely biochemical basis for the increased sensitivity of eggs to agonists, seen over time, that results in pronuclear formation. Also, all of these time-dependent changes caution against the use of mouse eggs > 16 h after hCG administration in studying the mechanism of normal fertilization and have implications for animal and human in vitro fertilization.
 
Article
Hyperactivated motility, a swimming pattern displayed by mammalian sperm in the oviduct around the time of ovulation, is essential to fertilization. Ca(2+) has been shown to be crucial for the initiation and maintenance of hyperactivated motility. Nevertheless, how Ca(2+) reaches the axoneme in the core of the flagellum to switch on hyperactivation is unknown. Ca(2+)-releasing agents were used to determine whether an intracellular store provides Ca(2+) to the axoneme. Hyperactivation was induced immediately in bull sperm by thapsigargin, caffeine, and thimerosal. The responses were dose-dependent and were induced in both capacitated and uncapacitated sperm. When external Ca(2+) was buffered below 50 nM with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, the response to caffeine was significantly reduced; however, the responses to thapsigargin and thimerosal were not affected. This indicates caffeine-induced hyperactivation depends on external Ca(2+) influx, whereas hyperactivation by thapsigargin and thimerosal do not. Acrosome reactions were not induced by these treatments; therefore, an acrosomal store was probably not involved. Indirect immunofluorescence labeling showed type I inositol 1,4,5-trisphosphate receptors (IP(3)R) in the acrosome and neck region, but no ryanodine receptors (RyR) were found using anti-RyR antibodies or BODIPY FL-X ryanodine. These data indicate that there is an IP(3)R-gated Ca(2+) store in the neck region of sperm that regulates hyperactivated motility.
 
Article
Germinal vesicle (GV)-intact fully grown mouse oocytes do not undergo cortical granule (CG) exocytosis in response to A23187 treatment, whereas metaphase II (MII)-arrested eggs do. This differential response may reflect the development of the ability of the egg to undergo CG exocytosis, which is responsible for the biochemical modification of the glycoprotein ZP2 in the zona pellucida. Accordingly, we compared in these two stages the ability of 12-O-tetradecanoyl phorbol 13-acetate (TPA) or inositol 1,4,5-trisphosphate (IP3) to promote CG exocytosis and/or the ZP2 to ZP2f conversion; these agents are known to stimulate early events of mouse egg activation. TPA (10 ng/ml) treatment for 60 and 120 min resulted in a 25% and 52% CG loss in GV-intact oocytes and a 38% and 76% loss in MII eggs, respectively; fertilization resulted in a CG loss of approximately 70-80%. Although a similar extent of ZP2 to ZP2f conversion was observed in oocytes and eggs after a 120-min TPA treatment (approximately 70-80%), a greater extent of conversion was observed in oocytes after a 60-min treatment (80% for oocytes, 50% for eggs). Microinjection of IP3 (final concentration 1 microM) into MII eggs resulted in an extent of ZP2 conversion similar to that observed following fertilization, whereas little conversion occurred in GV-intact oocytes similarly injected. These results indicate that a protein kinase C sensitivity develops prior to meiotic maturation, whereas responsiveness to IP3 develops after maturation has resumed. We propose that the regulatory mechanism involving an IP3-mediated calcium release is deficient in GV-stage oocytes.
 
A) Hoechst-stained rabbit MII oocyte at 14 h post-LH injection. The metaphase plate (MP) is located far from the first polar body (PB). B) Visualization of polar body (PB) and metaphase plate (MP) during oocyte enucleation by Hoechst staining and epifluorescence. C) Expanded NT blastocyst. D) Hatching NT blastocyst. 100. 
The effect of culture medium on the development of activated MII rabbit oocytes and in vivo-fertilized zygotes. 
The effect of activation treatments on parthenogenetic devel- opment of rabbit oocytes. 
Article
The present study was carried out to evaluate the effects of different activation protocols, enucleation methods, and culture media on the development of parthenogenetic and nuclear transfer (NT) rabbit embryos. Electroporation of 25 mM inositol 1,4, 5-trisphosphate (IP3) in calcium- and magnesium-free PBS immediately induced a single intracellular calcium transient in 6 out of 14 metaphase II-stage rabbit oocytes evaluated during a 10-min recording period. The percentage of oocytes treated with IP3 followed by 6-dimethylaminopurine (IP3 + DMAP) that cleaved (83.9%) and reached the blastocyst stage (50%) was significantly higher (p < 0.05) than those activated with multiple pulses (61.6% and 30.1%, respectively) or treated with ionomycin + DMAP (52.9% and 5.7%, respectively). Development of IP3 + DMAP-activated rabbit oocytes and in vivo-fertilized zygotes in different culture media was studied. Development of activated oocytes to the blastocyst stage in Earle's balanced salt solution (EBSS) supplemented with MEM nonessential amino acids, basal medium Eagle amino acids, 1 mM L-glutamine, 0.4 mM sodium pyruvate, and 10% fetal bovine serum (FBS) (EBSS-complete) (40.6%) was significantly higher (p < 0.05) than those that developed in either Dulbecco's Modified Eagle's medium (DMEM)/RPMI + 10% FBS (15.5%) or CR1aa + 10% FBS (4%) medium. In addition, 100% of in vivo-fertilized rabbit zygotes developed to the blastocyst stage in EBSS-complete. A third set of experiments was carried out to study the efficiency of blind versus stained (Hoechst 33342) enucleation of oocytes. Twenty-nine of 48 blind enucleated and IP3 + DMAP-activated oocytes cleaved (60.4%), and 15 (31.2%) subsequently reached the blastocyst stage, whereas 9 of 52 oocytes enucleated using epifluorescence (17.3%) cleaved, and none of these reached the blastocyst stage. When the above parameters that yielded the highest blastocysts were combined in an NT experiment using adult rabbit fibroblast nuclei, 72.2% (39 of 54) of the fused nuclear transplant embryos cleaved and 29.6% (16 of 54) reached the blastocyst stage.
 
Article
Precursor male gametes such as round spermatids and secondary spermatocytes are known to possess the potential to achieve fertilization and embryonic development when injected into mature oocytes, but in previous studies, injected mouse spermatids did not activate the oocytes. In this study, we confirmed that this was the case because spermatids by themselves could not induce an increase in intracellular Ca2+ concentration ([Ca2+]i) of oocytes, the pivotal signal in oocyte activation. Repetitive rises in [Ca2+]i (Ca2+ oscillations), lasting for at least 3 h as observed at fertilization, were produced by a single injection of adenophostin B isolated from fungal products, a novel nonmetabolizable agonist of the inositol 1,4,5-trisphosphate receptor (InsP3r), which mediates Ca2+ release from the endoplasmic reticulum. Ca2+ oscillations were blocked by an antibody against the type 1 InsP3r. About 95% of oocytes were activated by adenophostin (0.3-0.4 microM in the oocyte). Simultaneous injection of a round spermatid and adenophostin resulted in 55% fertilization success in association with male and female pronucleus formation and development to two-cell embryos. Furthermore, 25% of two-cell embryos that were transplanted to foster mothers developed to normal offspring. All infants grew into adults that reproduced healthy second generations. Adenophostin will be useful for parthenogenetic oocyte activation in the biotechnology of animal reproduction. Injection combined with precursor spermatozoa may be applicable to assisted conception therapy for patients with defective spermatogenesis.
 
Article
Mammalian fertilization is characterized by the presence of long-lasting intracellular calcium ([Ca2+]i) oscillations that are required to induce oocyte activation. One of the Ca2+ channels that may mediate this Ca2+ release is the inositol 1,4, 5-trisphosphate receptor (IP(3)R). Three isoforms of the receptor have been described, but their expression in oocytes and possible roles in mammalian fertilization are not well known. Using isoform-specific antibodies against IP(3)R types 1, 2, and 3 and Western analysis, we determined the isoforms that are expressed in bovine metaphase II oocytes and ovaries. In oocytes, all isoforms are expressed, but type 1 is present in overwhelmingly larger amounts and is likely responsible for the majority of Ca2+ release at fertilization. In ovarian microsomes, all three isoforms appear well expressed, suggesting the participation of all IP(3)R isoforms in ovarian Ca2+ signaling. We then investigated whether the reported cessation/reduction in amplitude of fertilization-associated [Ca2+]i oscillations, which is observed as pronuclear formation approaches, corresponded with down-regulation of the IP(3)R-1 isoform. Fertilization resulted in approximately 40% reduction in the amount of receptor by 16 h postinsemination. In addition, injection of adenophostin A, a potent IP(3)R agonist that elicits high-frequency [Ca2+]i oscillations in mammalian oocytes, induced similar reduction in receptor numbers. Together, these data show that 1) the three IP(3)R isoforms are expressed in bovine oocytes; 2) IP(3)R-1 is likely to mediate most of the Ca2+ release during fertilization; 3) its down-regulation may explain the decline in amplitude of sperm-induced [Ca2+]i rises as fertilization progresses toward pronuclear formation; and 4) agonists of the IP(3)R induce down-regulation of the type-1 receptor in oocytes similar to that evoked by fertilization.
 
Article
The mammalian X chromosome contains a large number of multicopy genes that are expressed during spermatogenesis. The roles of these genes during germ cell development and the functional significance of gene multiplication remain mostly unexplored, as the presence of multicopy gene families poses a challenge for genetic studies. Here we report the deletion of a 1.1 Mb segment of the mouse X chromosome that is syntenic with the human Xq22.1 region and contains 20 genes that are predominantly expressed in testis and brain, including 3 members of the nuclear export factor gene family (Nxf2, Nxf3, and Nxf7) and 5 copies of preferentially expressed antigen in melanoma-like 3 (Pramel3). We have shown that germline-specific Cre/loxP-mediated deletion of this 1.1 Mb segment is efficient and causes defective chromosomal synapsis, meiotic arrest, and sterility in male mice. Our results demonstrate that this 1.1 Mb region contains one or more novel X-linked factors that are essential for male meiosis.
 
Article
It was possible to demembrante and reactivate not only freshly collected testicular, cauda epididymal, and ejaculated ram sperm but also sperm that had been stored for several days at 0 degrees C and for several months at -196 degrees C in rete testis fluid or egg yolk citrate media. Sperm were usually washed free of seminal plasma before demembranation, but this was not essential for reactivation. Bovine serum albumin (1.0%) in the wash medium increased the survival of sperm, but more than 0.25% in the extraction medium decreased reactivation. A macro-molecular component of cauda epididymal fluid also inhibited the reactivation of testicular sperm. Triton X-100 concentrations between 0.01% and 1.00% in the extraction medium were satisfactory for demembranating the sperm. Rapid cooling (i.e., cold shock) mimicked the effect of detergent in making the sperm responsive to added ATP and demonstrated that damage to ram sperm in cold shock does not involve the axoneme. Ejaculated and cauda sperm were reactivated immediately on addition of ATP and activity persisted for up to 10 min. Testicular sperm, on the other hand, required about 4 min to become fully reactivated. The optimal ATP concentration for activation of sperm was 0.1-1.0 mM. Magnesium ions (0.1-1.0 mM) were important for reactivation, and testicular sperm required a higher magnesium concentration than did cauda or ejaculated sperm. Manganese ions were almost as effective as magnesium for reactivating cauda epididymal and ejaculated sperm. Cobalt and cadmium ions were much less active for cauda and ejaculated sperm and none of these ions were effective for testicular sperm. Fluoride (25-50 mM) inhibited reactivation. The presence of 50 microM cAMP in the extraction medium or preincubation of testicular sperm with theophylline or caffeine increased low levels of activation, but this was not evident with ejaculated or cauda sperm. We conclude that the motor apparatus is already functionally assembled in spermatozoa on leaving the testis, but some fine adjustment must take place during maturation in the epididymis.
 
Article
The purpose of this study was to characterize and quantify T lymphocyte populations and to investigate T cell antigen receptor (TcR) expression in human endometrium throughout the menstrual cycle and in early pregnancy. Frozen sections of endometrium were labeled using the highly sensitive avidinbiotin method and a panel of monoclonal antibodies. CD3-positive, CD8-positive, CD4-positive, TcR alpha beta-positive, and TcR gamma delta-positive cells were demonstrated in all phases of the menstrual cycle and in early pregnancy. There were no differences between pregnant and nonpregnant human endometrium in the proportion that each T cell subpopulation formed relative to the total CD3-positive T cell population. However, significantly fewer T cells were detected in early pregnant compared with nonpregnant endometrium. Our results clarify the TcR expression by T cells in pregnant and nonpregnant human endometrium and indicate that endometrial T cells are unlikely to play a significant role in implantation and the maintenance of human pregnancy since they decrease in number considerably in the first trimester of gestation.
 
Article
SDS-PAGE analysis of luminal fluid from the ram testis and epididymis revealed a protein of about 105 kDa in the fluid in the caput epididymal region. The molecular mass of this fluid protein shifted from 105 kDa to 94 kDa in the distal caput epididymidis and remained at 94 kDa in the lower regions of the epididymis. The possible sperm origin of this protein was suggested by the decrease in intensity of a 105-kDa compound on the sperm plasma membrane extract and by its total disappearance from the fluid of animals with impaired sperm production caused by scrotal heating. The 94-kDa protein was purified from ram cauda epididymal fluid, and a rabbit polyclonal antiserum was obtained. This antiserum showed that membranes of testicular sperm and sperm from the initial caput were positive for the presence of an immunologically related antigen. The protein was immunolocalized mainly on the flagellar intermediate piece, whereas in some corpus and caudal sperm, only the apical ridge of the acrosomal vesicle was labeled. The purified protein was microsequenced: its N-terminal was not found in the sequence database, but its tryptic fragments matched the sequence of the angiotensin I-converting enzyme (ACE). Indeed, the purified 94-kDa protein exhibited a carboxypeptidase activity inhibited by specific blockers of ACE. All the soluble seminal plasma ACE activity in the ram was attributable to the 94-kDa epididymal fluid ACE. The polyclonal antiserum also showed that a soluble form of ACE appeared specifically in the caput epididymal fluid of the boar, stallion, and bull. This soluble form was responsible for all the ACE activity observed in the fluid from the distal caput to the cauda epididymidis in these species. Our results strongly suggest that the epididymal fluid ACE derives from the germinal form of ACE that is liberated from the testicular sperm in a specific epididymal area.
 
Mir-17-92 (Mirc1) cluster miRNAs expressed in mouse testis. a) Schematic representation of the Mir-17-92 (Mirc1) cluster and its paralog Mir106b-25 (Mirc3) cluster. Pre-miRNAs are indicated as boxes, and mature miRNAs are represented by black boxes. The sequence of each mature miRNA is shown, and the miRNAs with the same seed sequence (italics and bold) are grouped and line coded. b) Northern blot analysis of the expression of the Mir17-92 (Mirc1) cluster in mouse testis. RNA from P19 cells was indicated as positive control. Equal RNA loading was confirmed by transfer RNA levels. Et, ethanol. c and d) In situ hybridization of Mir17 (c) and Mir20 (d) in the adult mouse testes. Cells with a positive signal are colored blue. Nucleus was counterstained with fast red. Red arrow, spermatogonia; green arrow, spermatocytes; black arrow, Sertoli cells; red arrowhead, interstitial cells. Bars ¼ 20 lm.
Retinoic acid signaling represses expression of the Mir-17-92 (Mirc1) and its paralog Mir-106b-25 (Mirc3) cluster miRNAs. a) Retinoic acidrepressed expression of the Mir-17-92 (Mirc1) cluster miRNAs in isolated THY1 þ spermatogonia. THY1 þ spermatogonia from six to eight mice at ages 6-8 days in each experiment were pooled and were treated with ethanol or RA (0.7 lM) for 24 h in vitro, and miRNAs were subjected to qRT-PCR. U6 snRNA was used for normalization between samples (mean 6 SD, *P , 0.05, ethanol control vs. RA treatment for 24 h, n ¼ 3; Student t-test). b) Treatment of VAD male mice with RA for 24 h significantly reduced the expression of the Mir17, Mir18a, Mir19a, and Mir19b in testes (mean 6 SD, *P , 0.05, sesame oil control vs. RA treatment for 24 h, n ¼ 8; Student t-test). c) Retinoic acid-repressed expression of the Mir-106b-25 (Mirc3) cluster miRNAs in isolated THY1 þ spermatogonia (mean 6 SD, *P , 0.05, ethanol control vs. RA treatment for 24 h, n ¼ 3; Student t-test). d) Treatment of VAD male mice with RA for 24 h significantly decreased the expression of the Mir-106b-25 (Mirc3) cluster miRNAs in testes (mean 6 SD, *P , 0.05, sesame oil control vs. RA treatment for 24 h, n ¼ 8; Student t-test).
a) Scheme of germ cell-specific Mir-17-92 (Mirc1) cluster knockout allele. Primers P1, P2, and P3 were used for genotyping. In germ cells, the Mir-17-92 (Mirc1) cluster will be deleted and thus allow for PCR amplification of a 441-bp product using P1 and P3. b) Genotyping PCR was performed on genomic DNA extracted from isolated THY1 þ enriched undifferentiated spermatogonia and tail from Mirc1 GCÀ/À (Mirc1 flox/flox ; Vasa-Cre), Mirc1 flox/flox , and Mirc1 flox/þ mice at 8–13 wk old. Notice both a Cre-deleted band (441 bp) and an intact floxed band (289 bp) presented in undifferentiated spermatogonia from Mirc1 GCÀ/À mice. Wild-type band: 255 bp. S, DNA extracted from isolated THY1 þ enriched undifferentiated spermatogonia; tail, DNA extracted from mouse tail. c) Quantitative RT-PCR analysis of the expression of the Mir-17-92 (Mirc1) cluster in germ cells from Mirc1 GCÀ/À and Mirc1 flox/flox mice. WT, Mirc1 flox/flox ; KO, Mirc1 GCÀ/À . Each experiment was performed in duplicate (n ¼ 4).  
Characterization of germ cell-specific Mir-17-92 (Mirc1) cluster knockout mice at ages 8–13 wk. a) Gross appearance of testes from Mirc1 GCÀ/À and its littermate Mirc1 flox/flox mice. Notice the markedly small testes in Mirc1 GCÀ/À mice. b and c) Testis weight and caudal epididymis sperm number of Mirc1 GCÀ/À and Mirc1 flox/flox mice. The relative testis weight (b) and caudal epididymis sperm number (c) of Mirc1 GCÀ/À mice are significantly decreased (mean 6 SD, *P , 0.01, n ¼ 4; Student t-test). d and e) Hematoxylin staining of transverse sections through the adult testes of Mirc1 GCÀ/À (e) and Mirc1 flox/flox (d) mice. Red arrow indicates the detached cells in seminiferous tubules, and asterisks represent the Sertoli cell-only tubules. Bars ¼ 20 lm.  
a) Quantitative RT-PCR analysis of the expression of Mir-106b-25 (Mirc3) cluster in germ cells from Mirc1 GCÀ/À and Mirc1 flox/flox mice at ages 813 wk. To yield enough RNA, individual RNA samples were extracted from three to five mice. Each experiment was performed in duplicate (mean 6 SD, *P , 0.05, n ¼ 4; Student t-test). b) Quantitative RT-PCR analysis of the transcription of Bcl2l11, Kit, Scos3, and Stat3 in germ cells from Mirc1 GCÀ/À and Mirc1 flox/flox mice (mean 6 SD, n ¼ 4).
Article
Increasing evidence indicates that microRNAs (miRNAs) may be critical players in spermatogenesis. The miRNA expression profiles of THY1(+)-enriched undifferentiated spermatogonia were characterized, and members of Mir-17-92 (Mirc1) and its paralog Mir-106b-25 (Mirc3) clusters are significantly downregulated during retinoic acid-induced spermatogonial differentiation, both in vitro and in vivo. The repression of microRNA clusters Mir-17-92 (Mirc1) and Mir-106b-25 (Mirc3) by retinoic acid in turn potentially upregulates the expression of Bim, Kit, Socs3, and Stat3. The male germ cell-specific Mir-17-92 (Mirc1) knockout mice exhibit small testes, a lower number of epididymal sperm, and mild defect in spermatogenesis. Absence of Mir-17-92 (Mirc1) in male germ cells dramatically increases expression of Mir-106b-25 (Mirc3) cluster miRNAs in the germ cells. These results suggest that Mir-17-92 (Mirc1) cluster and Mir-106b-25 (Mirc3) cluster miRNAs possibly functionally cooperate in regulating spermatogonial development.
 
PDC-109 promotes epididymal sperm binding to oviductal explants. Few epididymal sperm (EPD) bound to explants; however, addition of PDC-109 (EPD PDC) conferred on epididymal sperm the ability to bind explants to a degree comparable with that of washed ejaculated sperm (EJAC). Different letters denote significant differences among treatment groups (P 0.01). Means SEM are shown for four replicates.
Loss of PDC-109 during capacitation. Detergent extracts of 10 6 sperm were resolved by SDS-PAGE, blotted, and probed with anti-PDC109. Lane 1: 4 h in TALP (control); lane 2: 4 h in TALP heparin glucose (noncapacitated); lane 3: 4 h in TALP heparin (capacitated). 
Addition of PDC-109 to capacitated (CAP) sperm restored binding to the level observed for noncapacitated sperm (NON-CAP). Different letters denote significant differences among treatment groups (P 0.05). Means SEM are shown for three replicates. 
Article
Sperm reservoirs have been found in the oviducts of several species of mammals. In cattle, the reservoir is formed by the binding of sperm to fucose-containing glycoconjugates on the surface of oviductal epithelial cells. A fucose-binding molecule was purified from sperm extracts and identified as PDC-109 (BSP-A1/A2), a protein that is secreted by the seminal vesicles and associates with the plasma membrane of sperm upon ejaculation. The objective of this study was to demonstrate that PDC-109 promotes bull sperm binding to oviductal epithelium. PDC-109 was purified from bovine seminal plasma, and polyclonal antibodies were produced in rabbits. The antibodies detected PDC-109 on ejaculated sperm by indirect immunofluorescence and Western blots of extracts, but PDC-109 was not detected on epididymal sperm. When added to epididymal sperm, purified PDC-109 was absorbed onto the plasma membrane overlying the acrosome, as demonstrated by indirect immunofluorescence and by labeling sperm directly with fluorescein-conjugated PDC-109. When added to explants of oviductal epithelium, significantly fewer epididymal sperm than ejaculated sperm became bound. Addition of PDC-109 to epididymal sperm increased epithelial binding to the level observed for ejaculated sperm. In addition, binding of ejaculated sperm to oviductal epithelium was inhibited by addition of excess soluble PDC-109. Ejaculated sperm lost the ability to bind to oviductal epithelium after heparin-induced capacitation, but treatment with PDC-109 restored binding. These results demonstrate that PDC-109 enables sperm to bind to oviductal epithelium and plays a major role in formation of the bovine oviductal sperm reservoir.
 
Article
On ejaculation, sperm become coated with proteins secreted by the male accessory sex glands. In the bull, these proteins consist predominantly of the bovine seminal plasma family of proteins (BSPs): PDC-109 (BSP-A1/-A2), BSP-A3, and BSP-30-kDa. PDC-109 plays a role in forming an oviductal sperm reservoir by enabling sperm to bind to oviductal epithelium. Because PDC-109 has high sequence identity with the other BSPs, we tested BSP-A3 and BSP-30-kDa for the capacity to bind sperm to oviductal epithelium. BSP-A3 and BSP-30-kDa each increased binding of epididymal sperm to epithelium and were as effective as PDC-109 in competitively inhibiting binding of ejaculated sperm. Because binding extends the motile life of sperm, BSPs were tested for the ability to maintain sperm motility. BSP-treated epididymal sperm incubated with plasma membrane vesicles from bovine oviductal epithelium maintained progressive motility longer than untreated sperm. To our knowledge, this is the first report of this protective effect of BSPs. Similarities in function among the BSPs were reflected in their three-dimensional structure, whereas surface maps of electrostatic potential indicated differences in binding affinities and kinetics. Such differences may provide sperm with greater adaptability to variations among females. Altogether, these results indicate that BSPs play a crucial role in fertilization by maintaining sperm motility during storage.
 
Article
In order to map mouse tumor necrosis factor-alpha (TNF) gene expression in detail and to determine whether transcription or translation of the TNF gene is regulated by uterine colony stimulating factor-1 (CSF-1), preimplantation embryos, oviducts, uteri, and uteroplacental units were studied in various strains of mice. These included homozygous osteopetrotic (op/op) female mice, which completely lack CSF-1, and heterozygous (+/op) females, which have normal levels of CSF-1. TNF mRNA was identified in all samples except preimplantation embryos by use of Northern blot hybridization or reverse transcriptase polymerase chain reaction. In situ hybridization and immunocytochemical experiments showed that the TNF gene was expressed in mouse oviduct and uterine epithelial cells, decidual cells, macrophage-like cells, placental trophoblast, and embryos. Despite an absence of CSF-1, TNF gene expression in the uteri, placentas, and embryos of op/op mothers did not differ in any major respect from expression in +/op or other strains of mice. The results of this study therefore indicate that the TNF gene is transcribed and translated in an ordered sequence through mouse gestation, and that maternal CSF-1 is not essential to expression of this cytokine gene. Collectively, these findings are consistent with a major role for TNF in mouse reproduction and development and with a potential compensatory function for this potent polypeptide factor in CSF-1 deficiency.
 
TGF31 and-actin mRNAs were demonstrated in autoradiograms of Northern blots prepared from total RNA extracts of 51 endometrial tissue samples. Endometrial histology was classified as early, mid, and late proliferative (EP, MP, LP); early, mid, and late secretory (ES, MS, LS); and menstrual (M). Upper panel shows five representative samples. The lower panel shows the integrated optical density (IOD) data for TGFl31/p-actin mRNA. RNA prepared from a pool of endometrial stromal cells was used as an internal standard in each experiment (TGFI31/13-actin mRNA value = 1.00) to allow comparison between filters. The content of TGF[31 mRNA was higher (p = 0.003) in the later part (MS, LS, M) (n = 17) as compared to the early part of the cycle (EP, MP, LP, ES) (n = 34). 
Northern blot analysis of total RNA extracts of endometrial tissue explants obtained in the proliferative phase and treated under serum-free conditions in vitro for 1, 2, or 4 days with either estradiol + progesterone (solid bars) or ethanol for control (open bars). The number of experiments is given in the bars, and each experiment represents tissue from one patient. The IOD value for TGFI31 mRNA relative to that for-actin mRNA was calculated for each sample. The tissue content of TGF[31 mRNA was increased by treatment with estradiol + progesterone for 4 days (p = 0.005). 
Article
The purpose of this study was to identify cyclic variations and hormonal regulation of endometrial transforming growth factor beta1 (TGFbeta1) mRNA. Regulation of the plasminogen-activating system was also examined, since it is involved in activation of latent TGFbetas. We measured TGFbeta1 mRNA in 51 normal endometrial samples by Northern blot and densitometric scanning of autoradiograms. Each value was related to the corresponding beta-actin value to allow quantitative evaluation. TGFbeta1 mRNA was higher in the mid and late secretory and menstrual phases than in the earlier parts of the cycle. This pattern implies progesterone dependence. The content of TGFbeta1 mRNA in endometrial tissue explants obtained in the proliferative phase was significantly increased after stimulation for 4 days with estradiol + progesterone in vitro. Both TGFbeta1 and estradiol + progesterone increased the content of plasminogen activator inhibitor-1 mRNA and protein in primary cultures of endometrial stromal cells. Conditioned-medium concentrations of urokinase plasminogen activator (u-PA) were increased by TGFbeta1, but decreased by estradiol + progesterone. This effect of estradiol + progesterone results from increased internalization and degradation of u-PA secondary to up-regulation of the cell surface receptor for u-PA by progesterone (Casslén et al., JCEM 1995; 80: 2776-2784). Increased extracellular u-PA in response to TGFbeta1 exposure was thus in concordance with an unchanged amount of available u-PA receptors on the cell surface. The activation mechanism of latent TGFbeta involves u-PA activity; since u-PA activity is reduced in the secretory endometrium, we suggest that although TGFbeta1 mRNA is increased in the mid and late secretory phase, TGFbetas are mainly in their latent form until the premenstrual rise in u-PA activity stimulates activation. TGFbeta may promote capillary growth during endometrial regeneration.
 
Article
The 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) is responsible for the inactivation of glucocorticoids. This is the predominant isozyme in the human placenta, where it is proposed to protect the fetus from high levels of maternal cortisol. In the present study, we examined the effects of eicosanoids on the activity of 11beta-HSD2 in human choriocarcinoma JEG-3 cells, a well-established model for placental trophoblasts. Treatment of JEG-3 cells for 24 h with either prostaglandin (PG) E2 or F2alpha attenuated 11beta-HSD2 activity ( approximately 40%). Paradoxically, indomethacin, an inhibitor of cyclooxygenases, inhibited (approximately 40%) rather than stimulated the activity of this enzyme. This indicated that the arachidonic acid metabolism may be diverted to other pathway(s), the products of which may inhibit 11beta-HSD2 activity. To determine whether the lipoxygenase pathways were involved, the cells were treated with nordihydroguaretic acid (NDGA), a blocker of all three (5-, 12-, and 15-) lipoxygenases. NDGA caused a 3-fold increase in 11beta-HSD2 activity. To further delineate which specific lipoxygenase pathway was involved, the cells were incubated with zileuton, a selective inhibitor of 5-lipoxygenase. This resulted in a similar increase in 11beta-HSD2 activity, suggesting that the products of this pathway (e.g., leukotrienes) may be involved. Given that leukotriene B4 (LTB4) is the most biologically active product of the 5-lipoxygenase pathway, we treated the cells with LTB4, which inhibited 11beta-HSD2 activity in a time- and dose-dependent manner with a maximal effect (60% reduction) at 10 nM for 9 h. Semiquantitative reverse transcription-polymerase chain reaction analysis revealed that 11beta-HSD2 mRNA levels were not altered by the addition of LTB4, PGE2, or PGF2alpha, indicating an effect at the posttranscriptional level. In conclusion, these results demonstrate that prostaglandins and LTB4 are potent inhibitors of 11beta-HSD2 activity in JEG-3 cells, suggesting that placental 11beta-HSD2 activity is modulated by these locally produced eicosanoids. This is the first time that the products of arachidonic acid metabolism have been found to regulate the activity of 11beta-HSD2.
 
Article
Previous studies of the round goby (Neogobius melanostomus Pallas, 1814), an invasive fish species in the Laurentian Great Lakes of North America, have shown that this species has the ability to both synthesize and smell steroids that have a 5 beta-reduced and 3 alpha-hydroxyl (5 beta,3 alpha) configuration. An enzyme-linked immunoassay (EIA) for 3 alpha-hydroxy-5 beta-androstane-11,17-dione (11-O-ETIO) has been used to show a substantial rise in the rate of release of immunoreactive compounds into the water when males are injected with salmon gonadotropin releasing hormone analogue. Similar increases were noted for 11-ketotestosterone and 17,20 beta-dihydroxypregn-4-en-3-one. Partitioning of the extracts between diethyl ether and water showed the presence of both free and conjugated immunoreactive 11-O-ETIO. Only conjugated immunoreactivity was found in urine (implying that free steroid is released via the gills). The identity of the conjugates was probed by using HPLC, EIA, and mass spectrometry and removal of sulfate and glucosiduronate groups. Immunoreactivity in the conjugated fraction was found to be due mainly to 3 alpha,17beta-dihydroxy-5 beta-androstan-11-one 17-sulfate. However, the evidence was also strong for the presence in water extracts of substantial amounts of 3 alpha-hydroxy-5 beta-androstane-11,17-dione 3-glucosiduronate (which could be detected only by EIA after removal of the glucosiduronate group with beta-glucuronidase). There were also small amounts of 3 alpha-hydroxy-5 beta-androstane-11,17-dione 3-sulfate and 3 alpha,17beta-dihydroxy-5 beta-androstan-11-one 17-glucosiduronate. These studies give some idea of the types, amounts, and ratios of 11-O-ETIO derivatives that are released by reproductive N. melanostomus and will aid further research into the putative pheromonal roles of 5 beta,3 alpha-reduced androgens in this species.
 
Article
One hundred and thirteen crossbred gilts were used in three experiments to examine the relationship between the pattern or sequence of ovulation and subsequent variation in the morphology of Day 11 embryos. In the first experiment, the percentage of follicles that had ovulated was determined in individual gilts at 26, 30, 34, or 38 h after the onset of estrus (n = 20) and 39, 41, 43, 45, or 47 h post-injection of human chorionic gonadotropin (n = 25; hCG, 1000 IU). The second experiment consisted of observing the percentage of follicles ovulated in 52 additional gilts at 34 h after the onset of estrus (Day 0). In the third experiment, the morphological variation among littermate embryos was compared on Day 11 between sham-operated control gilts (n = 8) and gilts whose nonovulated follicles were destroyed by electrocautery (n = 8) on Day 1. Results of these experiments indicated that the pattern of ovulation in gilts was skewed (p less than 0.01). Ovulation, induced with hCG, appeared to occur in a majority of follicles during a short period of time, whereas the remaining ovulations occurred over a longer interval. Of the 57 gilts observed at 34 h after natural estrus, ovaries of 25 gilts contained corpora hemorrhagica (CH) and follicles; one gilt had 1 CH and 17 follicles, and 24 others had 10-17 CH with 1-4 follicles remaining. Destruction of these nonovulated follicles resulted in a more (p less than 0.01) uniform group of Day 11 embryos and with fewer (p less than 0.05) small embryos. These data demonstrated that the pattern of ovulation may affect morphological variation in embryonic development such that some of the later ovulating follicles may represent smaller embryos within a litter.
 
Article
Objectives were to examine possible roles of estrogen receptor (ER) in development of the bovine uterine endometrium in the context of ER type, enhancer type, and ligand-independent activation. Expression vectors producing either ERalpha or ERbeta were introduced into fetal uterine cells from Day 110 to 120 of gestation (UBF120 cells) and into rat embryo fibroblasts (Rat-1 cells), neither of which express endogenous ER. Reporter constructs containing either an estrogen response element (ERE) or activator protein-1 (AP-1) response element were cotransfected. These reporters were also transfected into fetal uterine cells from Day 180 to 200 of gestation (UBF180 cells), which express ER. In UBF120 and Rat-1 cells transfected with either ERalpha or ERbeta, treatment with estradiol-17beta (E2) resulted in increased activity of an ERE reporter construct, but not an AP-1 element reporter construct. The antiestrogen ICI 182,780 (ICI) exhibited E2 antagonist activity with both ERalpha and ERbeta. Thus, all components were present for E2-dependent transcription from an ERE except ER; however, cells were not competent for E2-dependent transcription mediated through AP-1. In UBF180 cells, E2 treatment increased both ERE and AP-1 reporter activity. ICI exhibited E2 antagonist activity. Treatment with epidermal growth factor resulted in increased ERE reporter activity that was inhibited by ICI, indicative of ligand-independent activation of ER. These data suggest that multiple pathways for ER-mediated gene regulation occur in the developing fetal uterus and that nuclear components necessary for action of both ERalpha and ERbeta are present prior to expression of the receptor.
 
TLR11 expression in the testis. A) Total RNA was extracted from the testis, kidney, and bladder of 5-wk-old mice. Relative Tlr11 mRNA level was determined using real-time qRT-PCR (left panel). TLR11 protein in tissue lysates was analyzed by Western blots using specific antibodies (right panel). B) TLR11 distribution in the testis. Immunohistochemistry was performed in paraffin sections of the testes of 5-and 2-wk-old mice. Insets in the upper right corners of the images are negative controls for immunostaining, in which the primary antibody was replaced by preimmune rabbit serum. The square areas of the left images were amplified for high resolution (right panels). Asterisk, interstitial cells; black arrow, round spermatid; black arrowhead, spermatocytes; white arrow, spermatogonia; white arrowhead, Sertoli cells; open arrow, elongating spermatid. Bar ¼ 20 lm. C) Testicular cell-specific expression. Testicular 
NFKB, IRF3, and MAPK activation. A) Profilin-induced NFKBp65 (p65) phosphorylation and IKB degradation. The germ cells of 5-wk-old mice were treated for the indicated durations. The cell lysates were analyzed by Western blots using specific antibodies against phosphor-p65 (p-p65), p65, and IKB; b-actin was used as loading control. B) NFKB, IRF3, and MAPK activation induced by UPEC. The germ cells were stimulated with 5 3 10 7 UPEC/ml for the indicated durations. NFKB and IRF3 activation were examined by analyzing p65 and IRF3 phosphorylation as well as IKB degradation using Western blot analysis (left panels). Activation of MAPKs, including p38, JNK, and ERK, was assessed by determining phosphor (p)-p38, p-JNK, and p-ERK via Western blot analysis using specific antibodies (right panels). Total p38, JNK, and ERK levels were used as controls. Images are representatives of at least three experiments using a germ cell pool of three mice. Data represent the mean 6 SEM of the three experiments. **P , 0.01. 
TLR11 intracellular localization and effect of bafilomycin (Baf) on innate immune response. A) TLR11 intracellular localization. Immunofluorescence staining was performed on frozen testicular sections 
TLR11 involvement in the innate immune response. A) Comparison of TLR11 expression in the germ cells of 5-and 2-wk-old 
Innate immune response of germ cells in vivo. Five-week-old male mice were used for in vivo study. One testis was injected with profilin or UPEC in PBS, the other one was injected with an equal volume of PBS and served as the control. A) NFKB, IRF3, and MAPK activation. Germ cells were enriched at 2 h after injection. The cell lysates were analyzed using Western blot for detecting p-p65, p65, and IKB (left panels) as well as p-IRF3/IRF3, pp38/p38, and p-JNK/JNK (right panels). B, C) Induction of cytokine expression. The germ cells were enriched at 4 h after injection with profilin (B) or UPEC (C). Total RNAs were extracted from the germ cells. Relative mRNA levels of the indicated cytokines were examined using real-time qRT-PCR. Western blot images are representatives of five experiments (one mouse each experiment). Data represent the mean 6 SEM values of five experiments. **P , 0.01. 
Article
Toxoplasma gondii and uropathogenic Escherichia coli (UPEC) may infect the testis and impair testicular function. Mechanisms underlying testicular innate immune response to these two pathogens remain to be clarified. The present study examined the function of TLR11, which can be recognized by T. gondii-derived profilin and UPEC, in initiating innate immune response in male mouse germ cells in vitro. TLR11 is predominantly expressed in spermatids. Profilin and UPEC induced the expressions of different inflammatory cytokine profiles in the germ cells. In particular, profilin induced the expressions of macrophage chemotactic protein 1 (MCP-1), interleukin (IL)-12 and interferon (IFN)-γ through nuclear factor κB (NF-κB) activation. UPEC induced the expressions of MCP-1, IL-12, and IFN-γ, as well as tumor necrosis factor alpha (TNF-α), IL-6, and IFN-β, through the activation of NF-κB, IFN regulatory factor 3 and mitogen-activated protein kinases. Evidence showed that profilin induced the innate response in male germ cells through TLR11 signaling, and UPEC triggered the response through TLR11 and other TLR signaling pathways. We also provided evidence that local injection of profilin or UPEC induces the innate immune response in the germ cells. Data describe TLR11-mediated innate immune function of male germ cells in response to T. gondii profilin and UPEC stimulations in vitro. This system may play a role in testicular defense against T. gondii and UPEC infections in mice.
 
Microarray analysis from the penis (A) and testis (B) at 7 days of age in rats treated from 1 to 6 days of age with DES. Compared to controls, note in the penis 60%-90% reduction in gene expression profiles of markers for smooth muscle cell or angiogenesis and, in the testis, 80%95% reduction in gene expression profiles of steroidogenic enzymes.
Article
Cavernous smooth muscle cells are essential components in penile erection. In this study, we investigated effects of estrogen exposure on biomarkers for smooth muscle cell differentiation in the penis. Neonatal rats received diethylstilbestrol (DES), with or without the estrogen receptor (ESR) antagonist ICI 182,780 (ICI) or the androgen receptor (AR) agonist dihydrotestosterone (DHT), from postnatal days 1-6. Tissues were collected at 7, 10, or 21 days of age. Smooth muscle cell biomarker MYH11 was studied in-depth because microarray data showed its significant down-regulation, along with other biomarkers, in DES treatment. Quantitative RT-PCR and Western blot analyses showed 50-80% reduction (P≤0.05) in MYH11 expression in DES-treated rats, compared to controls; and ICI and DHT co-administration mitigated the decrease. Temporally from 7-21 days of age, Myh11 expression increased one-fold (P≥0.05) in DES-treated rats versus three-fold (P≤0.001) in controls, implying a long-lasting inhibitory effect of DES on smooth muscle cell differentiation. Immunohistochemical localization of smooth muscle alpha actin, another biomarker for smooth muscle cell differentiation, showed fewer cavernous smooth muscle cells in DES-treated animals, compared to controls. Additionally, DES treatment significantly up-regulated Esr1 mRNA expression, and suppressed the neonatal testosterone surge by 90%, which was mitigated by ICI co-administration but not by DHT co-administration. Collectively, results provided evidence that DES treatment to neonatal rats inhibited cavernous smooth muscle cell differentiation, as shown by down-regulation of MYH11 expression at the mRNA and protein levels and by reduced immunohistochemical staining of smooth muscle alpha actin. Both ESR and AR pathways probably mediate this effect.
 
Article
Successful pregnancy depends on the precise regulation of extravillous trophoblast (EVT) invasion into the uterine decidua, primarily by decidua-derived factors. In humans, during early pregnancy interleukin 11 (IL11) is maximally expressed in the decidua, with its receptor, IL11 receptor alpha (IL11RA), also identified on invasive EVTs in vivo. Although a role for IL11 in EVT migration has been established, whether it also plays a role in regulating EVT invasion is unknown. We investigated whether IL11 influences human EVT invasion and the signaling pathways and underlying mechanisms that may be involved, using the HTR-8/SVneo immortalized EVT cell line and primary EVTs as models for EVTs. Interleukin 11 (100 ng/ml) significantly inhibited invasion of EVT cells by 40% to 60% (P < 0.001). This effect was abolished by inhibitors of signal transducer and activator of transcription 3 (STAT3) but not of mitogen-activated protein kinase (MAPK) pathways. Interleukin 11 (100 ng/ml) had no effect on matrix metallopeptidases 2 and 9 (MMP2 and MMP9), tissue inhibitors of MMP (TIMP1, TIMP2, and TIMP3), plasminogen activator urokinase (PLAU), plasminogen activator urokinase receptor (PLAUR), and serpin peptidase inhibitors 1 and 2 (SERPINE1 and SERPINE2) in EVT-conditioned media and/or cell lysates. Interleukin 11 (100 ng/ml) also did not regulate EVT cell adhesion or integrin expression. These data demonstrate that IL11 inhibits human EVT invasion via STAT3, indicating a likely role for IL11 in the decidual restraint of EVT invasion during normal pregnancy.
 
Effects of breed, uterine environment, and gestational age on placental NAD -dependent 11HSD dehydrogenase activity under optimal in vitro conditions of pH (9.0), coenzyme concentration (800 M), substrate concentration (2.5 M plus 0.86 Ci [ 3 H]cortisol), time (15 min), and temperature (37C). Each bar represents the mean SEM of the number of pigs in parentheses. Statistics were conducted on squareroot-transformed data. A priori orthogonal contrasts show that this enzymatic activity was greater in Meishan placenta compared with WC-INT placenta on Day 24 (P 0.037, a vs. b). WC-INT, intact white crossbred gilts; WC-UHO, unilaterally-hysterectomized-ovariectomized white crossbred gilts; ME, intact Meishan gilts.  
A) Northern blot analysis of placental GR mRNA expression at Days 24, 30, and 40 of gestation. Each lane contained 30 g of total RNA. B) Results of densitometric analysis (Epi Chemi Darkroom; UVP, Inc., Upland, CA) of steady-state levels of placental GR mRNA expression. Values for GR were adjusted by analysis of covariance to those of -actin for the same sample. Results represent the least-squares mean SEM of the number of replicates indicated by numbers within bars. Statistics were conducted on log 10 -transformed data. WC-INT, intact white crossbred gilts; WC-UHO, unilaterally-hysterectomized-ovariectomized white crossbred gilts; ME, intact Meishan gilts.  
A) Northern blot analysis of placental 11HSD-1 mRNA expression at Days 24, 30, and 40 of gestation. Each lane contained 10 g of total RNA. B) Results of densitometric analysis (ChemImager; Alpha Innotech Corp., San Leandro, CA) of steady-state levels of placental 11HSD-1 mRNA. Values for 11HSD-1 were adjusted by analysis of covariance to those of -actin for the same sample. Results represent the least-squares mean SEM of the number of replicates indicated by numbers within bars. WC-INT, intact white crossbred gilts; WC-UHO, unilaterally-hysterectomized-ovariectomized white crossbred gilts; ME, intact Meishan gilts.  
A) Northern blot analysis of placental 11HSD-2 mRNA expression at Days 24, 30, and 40 of gestation. Each lane contained 10 g of total RNA. B) Results of densitometric analysis (ChemImager; Alpha Innotech Corp., San Leandro, CA) of steady-state levels of placental 11HSD-2 mRNA for only the major band in lane A. Values for 11HSD- 2 were adjusted by analysis of covariance to those of -actin for the same sample. Results represent the least-squares mean SEM of the number of replicates indicated by numbers within bars. Statistics were conducted on log 10 -transformed data. A priori orthogonal contrasts indicate that on Day 30, 11HSD-2 mRNA expression is less in WC-UHO pigs than in WC-INT pigs (P 0.03, a vs. b). WC-INT, intact white crossbred gilts; WC-UHO, unilaterally-hysterectomized-ovariectomized white crossbred gilts; ME, intact Meishan gilts.  
Article
Glucocorticoids are known to influence many aspects of prenatal development. Three important regulators of glucocorticoid actions at the cellular level are the enzymes 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD-1), 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD-2), and glucocorticoid receptors (GR). The present study was conducted to determine the presence of these regulators in porcine placentae during early gestation (Days 24-40; term = 114 days) and to examine the influence of breed and uterine environment. Three pig models differing in uterine environment as reflected by embryonic survival from Days 24 to 40 were used: intact white cross-bred gilts (WC-INT); white cross-bred gilts that had been unilaterally hysterectomized-ovariectomized before puberty (WC-UHO); and intact Meishan gilts (ME). Porcine-specific partial cDNAs for 11betaHSD-1 and 11betaHSD-2 and a cRNA for GRalpha were developed and used to produce 32P-labeled probes for Northern blot analyses. The 11betaHSD dehydrogenase activity was measured in vitro at saturating concentrations of substrate and coenzyme. At Day 24 of gestation, 11betaHSD-2 mRNA, dehydrogenase activity, and GR mRNA were present, but 11betaHSD-1 mRNA was absent. All three mRNAs and dehydrogenase activity increased (P < 0.01) by Day 40. On Day 30, placental 11betaHSD-2 mRNA was decreased (P = 0.03) by 47% in WC-UHO versus WC-INT. Placental 11betaHSD dehydrogenase activity was 2-fold greater (P < 0.01) in ME versus WC-INT on Day 24 of gestation. These results demonstrate, to our knowledge for the first time, the presence of 11betaHSD-1, 11betaHSD-2, and GR mRNA as well as 11betaHSD dehydrogenase activity in the porcine placenta during early pregnancy. Moreover, a role for glucocorticoids in porcine embryonic development is suggested.
 
Article
Poliovirus receptor (Pvr), erythrocyte protein band 4.1-like 3 (Epb4.1l3), regulator of G-protein signaling 11 (Rgs11), and oxytocin receptor (Oxtr) expression were quantified in in vitro- and in vivo-grown mouse follicles. The expression of all genes was increased during antral growth in in vitro-grown cumulus cells, whereas only Rgs11 and Oxtr were increased and Pvr and Epb4.1l3 were decreased in in vivo grown cumulus cells. In vivo mural granulosa cells showed the highest expression of Pvr, Rgs11, and Oxtr. The in vitro granulosa + theca compartment responded to human chorionic gonadotropinduring early luteinization by either an upregulation (Pvr, Oxtr) or downregulation (Epb41l3, Rgs11). Oocytes expressed Epb4.1l3, not Rgs11, and Pvr only in in vitro-grown oocytes. Translation into protein was confirmed for Epb4.1l3 in in vitro-grown follicles and in vivo-grown cumulus-oocyte complexes. Protein 4.1B was present during antral growth in cumulus, granulosa cells, and oocytes. Hypothetical functions of Epb4.1l3 and Pvr involve cell adhesion regulation and Rgs11 could be involved in cAMP production in the follicle. Oxtr is known to be important during and after the ovulatory stimulus, but, as in bovine, was also regulated during folliculogenesis. High expression of Pvr and Epb4.1l3 with culture duration in cumulus cells might mark inappropriate differentiation into a mural granulosa-like cell type and function as negative follicle development marker. Rgs11 and Oxtr are both in vivo and in vitro upregulated in cumulus cells during antral follicle growth and might be considered positive markers for follicle development.
 
Top-cited authors
Pat Lonergan
  • University College Dublin
Thomas E Spencer
  • Washington State University
Fuller W Bazer
  • Texas A&M University
I. Martin Sheldon
  • Swansea University
Niamh Forde
  • University of Leeds