A gradual alteration in the mechanisms underlying reproduction and fertility characterizes the aging process in human females. These changes culminate in menopause, conventionally defined as a cessation of menstrual cycles that marks the end of reproductive capacity. In fact, a central and defining event in menopause is the discontinuation of ovulation, which is correlated with a number of structural and functional changes in the reproductive axis. Despite several decades of research, a degree of uncertainty remains as to whether nonhuman primates undergo menopause, and whether they are suitable models of human reproductive senescence. We review some of the controversies that have clouded our understanding of reproductive aging in nonhuman primates, including issues of definition, timing, comparability of data from wild versus captive populations, and cross-species comparisons. The existing data support the view that menopause occurs in a number of primate species and is not unique to humans.

A long postreproductive lifespan may distinguish women from all other female primates. A long-held consensus among reproductive scientists has been that our closest living relative, the chimpanzee (Pan troglodytes), experiences menstrual cycles until death. However, a recent study of biannual assessments of gonadotropins, but lacking observations of menstruation, concluded that menopause occurs in chimpanzees between 35 and 40 yr of age. A separate report, but on wild chimpanzees, documented fertility through the 40–44 age range in all populations studied. These contradictory reports pose questions about differences between wild and captive populations and about assessments of menopause. The present study revisits this controversy by analyzing longitudinal records of anogenital swelling and menstruation in 89 female chimpanzees aged 6 to 59 yr (n = 2386 records on cycle length), monitored for most of their adult lives at the Yerkes National Primate Research Center. Twenty of these chimpanzees were observed past 39 yr of age; all 20 displayed menstrual cycles beyond this age, as confirmed by at least two observations of menses about 35 days apart. Three of these were older than 50 yr and still displayed menstrual cycles. Only the oldest female appeared menopausal, with cycles of anogenital swelling ceasing 2 yr prior to her death at age 59. Random-effects statistical modeling reveals a slight decrease in cycle length until 20 yr of age and a slight lengthening thereafter. Mean cycle length across the lifespan is 35.4 days. Our findings, based upon actual observations of menstrual cycles, suggest that menopause in the chimpanzee is rare, occurring near the end of the lifespan.

Tissue-specific novel transcripts expressed during sexual development were examined by RT-PCR, quantitative RT-PCR (qRT-PCR), and in situ hybridization to provide data for chicken genomics. Public databases for transcript data have been constructed with known and unknown sequences of various tissues from different animals. However, the expression patterns and functions of the transcripts are less known. From the The Institute for Genomics Research Gallus gallus library, we examined 291 tentative consensus (TC) sequences that assembled 100% with transcripts by RT-PCR during male and female sexual development from Embryonic Day 6 to 25 wk of age. We found 85 TC sequences that were specific to testicular development; of these, 43 TC sequences were exclusively upregulated in 25-wk-old testis. Another 52 TC sequences were not specific to one tissue, but occurred in the testis and ovary at different developmental ages. Twelve testis-specific TC sequences upregulated in 25-wk-old testis were randomly selected and further examined with qRT-PCR. For precise localization, these 12 testis-specific TC sequences were examined by in situ hybridization with 25-wk-old adult testis. Six TC sequences were strongly expressed in secondary spermatocytes and haploid spermatids until spermatozoa release. Another six TC sequences were differentially expressed in the adluminal compartment of seminiferous tubules. Among the testis-specific TC sequences, TC120901 is a known gene, phospholipase C, zeta (PLCZ1). Our data provide potential insight into gene expression and genomic information on novel transcripts that are important to avian reproduction.

The equatorial subsegment (EqSS) was originally identified by atomic force microscopy as a discrete region within the equatorial segment of Artiodactyl spermatozoa. In this investigation, we show that the EqSS is enriched in tyrosine phosphorylated proteins and present preliminary evidence for its presence in mouse and rat spermatozoa. The anti-phosphotyrosine monoclonal antibody (McAb) 4G10 bound strongly and discretely to the EqSS of permeabilized boar, ram, and bull spermatozoa. It also bound to a small patch on the posterior acrosomal region of permeabilized mouse and rat spermatozoa, suggesting that the EqSS is not restricted to the order Artiodactyla. An anti-HSPA1A (formerly Hsp70) antibody recognized the EqSS in boar spermatozoa. Immunogold labeling with McAb 4G10 localized the tyrosine phosphorylated proteins to the outer acrosomal membrane. This was verified by freeze-fracture electron microscopy, which identified the EqSS in three overlying membranes, the plasma membrane, outer acrosomal membrane, and inner acrosomal membrane. In all five species, tyrosine phosphorylated proteins became restricted to the EqSS during sperm maturation in the epididymis. The major tyrosine phosphorylated proteins in the EqSS of boar and ram spermatozoa were identified by mass spectrometry as orthologs of human SPACA1 (formerly SAMP32). Immunofluorescence with a specific polyclonal antibody localized SPACA1 to the equatorial segment in boar spermatozoa. We speculate that the EqSS is an organizing center for assembly of multimolecular complexes that initiate fusion competence in this area of the plasma membrane following the acrosome reaction.

The efferent ductules express the highest amount of estrogen receptors ESR1 (ERalpha) and ESR2 (ERbeta) within the male reproductive tract. Treatment of rats with the antiestrogen fulvestrant (ICI 182,780) causes inhibition of fluid reabsorption in the efferent ductules, leading to seminiferous tubule atrophy and infertility. To provide a more comprehensive knowledge about the molecular targets for estrogen in the rat efferent ductules, we investigated the effects of ICI 182,780 treatment on gene expression using a microarray approach. Treatment with ICI 182,780 increased or reduced at least 2-fold the expression of 263 and 98 genes, respectively. Not surprisingly, several genes that encode ion channels and macromolecule transporters were affected. Interestingly, treatment with ICI 182,780 markedly altered the expression of genes related to extracellular matrix organization. Matrix metalloproteinase 7 (Mmp7), osteopontin (Spp1), and neuronal pentraxin 1 (Nptx1) were among the most altered genes in this category. Upregulation of Mmp7 and Spp1 and downregulation of Nptx1 were validated by Northern blot. Increase in Mmp7 expression was further confirmed by immunohistochemistry and probably accounted for the decrease in collagen content observed in the efferent ductules of ICI 182,780-treated animals. Downregulation of Nptx1 probably contributed to the extracellular matrix changes and decreased amyloid deposition in the efferent ductules of ICI 182,780-treated animals. Identification of new molecular targets for estrogen action may help elucidate the regulatory role of this hormone in the male reproductive tract.

Lhx8 is a member of the LIM-homeobox transcription factor family and preferentially expressed in oocytes and germ cells within the mouse ovary. We discovered that Lhx8 knockout females lose oocytes within 7 days after birth. At the time of birth, histological examination shows that Lhx8-deficient (Lhx8^−/−) ovaries are grossly similar to the newborn wild-type ovaries. Lhx8^−/− ovaries fail to maintain the primordial follicles, and the transition from primordial to growing follicles does not occur. Lhx8^−/− ovaries misexpress oocyte-specific genes, such as Gdf9, Pou5f1, and Nobox. Very rapid loss of oocytes may partly be due to the drastic downregulation of Kit and Kitl in Lhx8^−/− ovaries. We compared Lhx8^−/− and wild-type ovaries using an Affymetrix 430 2.0 microarray platform. A total of 80 (44%) of 180 of the genes downregulated more than 5-fold in Lhx8^−/− ovaries were preferentially expressed in oocytes, whereas only 3 (2%) of 146 genes upregulated more than 5-fold in the absence of Lhx8 were preferentially expressed in oocytes. In addition, the comparison of genes regulated in Lhx8^−/− and Nobox^−/− newborn ovaries discovered a common set of 34 genes whose expression level was affected in both Lhx8- and Nobox-deficient mice. Our findings show that Lhx8 is a critical factor for maintenance and differentiation of the oocyte during early oogenesis, and it acts in part by downregulating the Nobox pathway.

Transient, human and murine decidua-associated, Natural Killer lymphocytes (uNK cells) have special, localized roles in early gestational endometrial remodeling and angiogenesis. To determine if uNK cells promote a specific vessel subtype, a histological time-course study of implantation site endothelia was undertaken using normal C57BL/6J (B6) and uNK-deficient B6.129-Rag2^tm1FwaIl2rg^tm2Cgn (alymphoid) mice, a strain lacking pregnancy-induced structural modifications of spiral arteries. Antibodies to EFNB2, EPHB4, and LYVE1, respectively, identified arterial, venous, and lymphatic endothelia. Unexpectedly, many uNK cells in B6 endometrium showed strong EFNB2 expression early in gestation, then became EPHB4 . This molecular transition coincided with structural modifications of spiral arteries that shifted from EFNB2 /EPHB4⁻ to EFNB2 /EPHB4 . NK cells from B6 spleen and liver did not express EFNB2. LYVE1 expression was similar in endometrium from B6 and alymphoid mice, but EFNB2 and EPHB4 expression in alymphoid mice was dramatically different. Strong stromal expression of both molecules developed mesometrially, and this was reduced by B6 lymphocyte transfer. Trophoblasts reacted with each marker in both strains. Expression of EFNB2 by uNK cells and trophoblasts may be the key regulatory mechanism that drives their positional association with EFNB2 arteries and prevents association of both cell types with EPHB4 veins. Gain of EPHB4 by midgestation spiral arteries may signal completion of pregnancy-induced arterial modification and provide a repulsion mechanism that limits subsequent interactions of the modified vessel with uNK cells and trophoblasts.

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.

Reproductive capacity is fundamental to the survival of all species. Consequently, much research has been undertaken to better understand gametogenesis and the interplay between germ cells and the somatic cell lineages of the gonads. In this study, we have analyzed the embryonic expression pattern of the X-linked gene family Reproductive homeobox genes on the X chromosome (Rhox) in mice. Our data show that eight members of the Rhox gene family are developmentally regulated in sexually dimorphic and temporally dynamic patterns in the developing germ cells during early gonadogenesis. These changes coincide with critical stages of differentiation where the germ cells enter either mitotic arrest in the testis or meiotic arrest in the ovary. Finally, we show that Rhox8 (Tox) is the only member of the Rhox gene family that is expressed in the somatic compartment of the embryonic gonads. Our results indicate that the regulation of Rhox gene expression and its potential function during embryogenesis are quite distinct from those previously reported for Rhox gene regulation in postnatal gonads.

WNT/CTNNB1 signaling is involved in the regulation of multiple embryonic developmental processes, adult tissue homeostasis, abd cell fate determination and differentiation. Many WNTs and components of the WNT/CTNNB1 signaling pathway are expressed in the testis, but their physiological roles in this organ are largely unknown. To elucidate the role(s) of WNT/CTNNB1 signaling in the testis, transgenic Ctnnb1^tm1Mmt/ ;Amhr2^tm3(cre)Bhr/ mice were generated to obtain sustained activation of the WNT/CTNNB1 pathway in both Leydig and Sertoli cells. Male Ctnnb1^tm1Mmt/ ;Amhr2^tm3(cre)Bhr/ mice were sterile because of testicular atrophy starting at 5 wk of age, associated with degeneration of seminiferous tubules and the progressive loss of germ cells. Although Cre activity was expected in Ctnnb1^tm1Mmt/ ;Amhr2^tm3(cre)Bhr/ Leydig cells, no evidence of Cre-mediated recombination of the floxed allele or of WNT/CTNNB1 pathway activation could be obtained, and testosterone levels were comparable to age-matched controls, suggesting that genetic recombination was inefficient in Leydig cells. Conversely, sustained WNT/CTNNB1 pathway activation was obtained in Ctnnb1^tm1Mmt/ ;Amhr2^tm3(cre)Bhr/ Sertoli cells. The latter often exhibited morphological characteristics suggestive of incomplete differentiation that appeared in a manner coincident with germ cell loss, and this was accompanied by an increase in the expression of the immature Sertoli cell marker AMH. In addition, a poorly differentiated, WT1-positive somatic cell population accumulated in multilayered foci near the basement membrane of many seminiferous tubules. Together, these data suggest that the WNT/CTNNB1 pathway regulates Sertoli cell functions critical to their capacity to support spermatogenesis in the postnatal testis.

Tetraploid (4n) embryo complementation assay has shown that embryonic stem (ES) cells alone are capable of supporting embryonic development (ES mouse), allowing the recovery of mouse lines directly from cultured ES cell lines. Although the advantages of this technique are well recognized, it remains inefficient for generating ES mice. In the present study, we investigated the effects of cell number of host 4n embryos on the production of ES mice. Four independent ES cell lines (two general ES cell lines and two nuclear transfer-derived ES cell lines) were used, and each cell line was aggregated with single (1×) to triple (3×) host 4n embryos. We found that birth rate of ES mice using 1× 4n embryos was quite low (0–2%) regardless of cell line, whereas except for one cell line, approximately 6–14% of embryos developed to full term in the case of 3× 4n embryos. Contamination of host 4n cells in ES mice was quite rare, being comparable to that generated using general methods even if they were delivered from 3× 4n host embryos. These results demonstrate that the use of 3× 4n embryos is effective for generating ES mice. Our technique described here will be applicable to any ES cell line, including general ES cell lines used for gene targeting.

Caltrin is a small and basic protein of the seminal vesicle secretion that inhibits sperm calcium uptake. The influence of rat caltrin on sperm physiological processes related to fertilizing competence was studied by examining its effect on 1) spontaneous acrosomal exocytosis, 2) protein tyrosine phosphorylation, and 3) sperm-egg interaction. Results show that the presence of caltrin during in vitro capacitation both reduced the rate of spontaneous acrosomal exocytosis without altering the pattern of protein tyrosine phosphorylation, and enhanced the sperm ability to bind to the zona pellucida (ZP). The significantly higher proportion of sperm with intact acrosome observed in the presence of caltrin was accompanied by a strong inhibition in the acrosomal hyaluronidase release. Enhancement of sperm-ZP binding was evident by the increase in the percentage of eggs with bound spermatozoa as well as in the number of bound sperm per egg. Similar results were obtained when the assays were performed using spermatozoa preincubated with caltrin and then washed to remove the unbound protein, indicating that the sperm-bound caltrin was the one involved in both acrosomal exocytosis inhibition and sperm-ZP binding enhancement. Caltrin bound to the sperm head was partially released during the acrosomal exocytosis induced by Ca-ionophore A23187. Indirect immunofluorescence and immunoelectron microscopy studies revealed that caltrin molecules distributed on the dorsal sperm surface disappeared after ionophore exposure, whereas those on the ventral region remained in this localization after the treatment. The present data suggest that rat caltrin molecules bound to the sperm head during ejaculation prevent the occurrence of the spontaneous acrosomal exocytosis along the female reproductive tract. Consequently, more competent spermatozoa with intact and functional acrosome would be available in the oviduct to participate in fertilization.

Epithelia lining the male reproductive duct modulate fertility by altering the luminal environment to which sperm are exposed. Although vas deferens epithelial cells reportedly express high levels of cyclooxygenases (Ptgs), and activation of bradykinin (BK) receptors can lead to upregulation of PTGS activity in epididymal epithelia, it remains unknown whether BKs and/or PTGSs have any role in modulating epithelial ion transport across vas deferens epithelia. Porcine and human vas deferens epithelial cell primary cultures and the PVD9902 cell line responded to lysylbradykinin with an increase in short circuit current (I_SC; indicating net anion secretion), an effect that was 60%–93% reduced by indomethacin. The BK effect was inhibited by the B2 receptor subtype (BDKRB2) antagonist HOE140, whereas the B1 receptor subtype agonist des-Arg⁹-BK had no effect. BDKRB2 immunoreactivity was documented in most epithelial cells composing the native epithelium and on Western blots derived from cultured cells. Gene expression analysis revealed that the PTGS2 transcript is 20 times more abundant than its PTGS1 counterpart in cultured porcine vas deferens epithelia and that BDKRB2 mRNA is likewise highly expressed. Subsequent experiments revealed that prostaglandin E2, 1-OH prostaglandin E1 (prostaglandin E receptor 4 [PTGER4] agonist) and butaprost (PTGER2 agonist) increase I_SC in a concentration-dependent manner, whereas sulprostone (mixed PTGER1 and PTGER3 agonist) produced no change in I_SC. These results demonstrate that autacoids can affect epithelial cells to acutely modulate the luminal environment to which sperm are exposed in the vas deferens by enhancing PTGS activity, leading to the production of prostaglandins that act at PTGER4 and/or PTGER2 to induce or enhance anion secretion.

In the gastrointestinal tract, interstitial cells of Cajal (ICCs) generate a pacemaker activity. They produce electric slow waves that trigger and coordinate gut smooth muscle contractions. Interstitial cells of Cajal's slender shape is revealed by KIT immunostaining. Based on several features, including KIT expression and KIT dependence, ICC-like cells were identified in nongastrointestinal tissues. Here, we investigated in the mouse whether uterine contractions depend on ICC-like cells' activity. By labeling KIT-expressing cells, we found putative ICC-like cells in the uterus, observed as KIT-positive interstitial, long spindle-shaped cells with fine branched cytoplasm processes, distributed in muscular layers and in subepithelial connective tissue. We then checked the potential KIT dependence of ex vivo contractile activity of the uterus by combining genetic and pharmacological approaches, using the Kit^W-v hypomorphic mutation, and imatinib as a KIT noncompetitive inhibitor. We found a significant reduction in frequency of longitudinal uterine contractions in Kit^W-v/Kit^W-v compared with Kit ^/ mice, whereas amplitude was unaffected. There was no difference in frequency or amplitude of circular uterine contractions between Kit^W-v/Kit^W-v and Kit ^/ mice. Ex vivo treatment of Kit ^/ uterine horns with imatinib resulted in a dose-dependent reduction of the frequency and amplitude of longitudinal myometrial contractions. Amplitude and frequency of circular contractions were unaffected in presence of imatinib. These concurrent results suggest that longitudinal contractions of the uterus depend on a KIT signaling pathway of ICC-like cells. The existence of ICC-like cells in the myometrium may enhance our understanding of uterine spontaneous contractile activity and suggest new approaches for treatment of uterine contractility disorders.

Interferon-tau (IFNT) is secreted by the conceptus trophoblast and signals pregnancy recognition in ruminants. IFNT regulates expression of genes in the endometrium, peripheral blood leukocytes (PBLs), and corpus luteum (CL). Microarray analysis identified that expression of (chemosensory) receptor transporter protein 4 (RTP4) increased in PBLs during early pregnancy in cows. In the present study, we cloned and characterized RTP4 transcription during early pregnancy in ewes. Endometrium, PBLs, and CL were collected on Days 11, 13, and 15 of the cycle and on Days 11, 13, 15, 17, and 19 of pregnancy. Northern blot analysis revealed an expected 1.6-kb mRNA and an unexpected 2.6-kb mRNA. In endometria, RTP4 mRNA levels in cyclic ewes remained low, whereas RTP4 mRNA increased from Day 11 to Day 17 in pregnant ewes. Levels of RTP4 mRNA also increased from Day 15 to Day 19 in CL and PBL samples from pregnant ewes only. The RTP4 mRNA was located in the glandular epithelium, stratum compactum, and caruncular stroma. Ovine glandular epithelial cells were treated with IFNT to determine if IFNT alone could induce RTP4. IFNT increased RTP4 more than 70-fold at 1.5 h after treatment, with maximal induction of nearly 300-fold above values observed in nontreated controls at 6 h after treatment. These results indicate that RTP4 mRNA levels are induced in the ovine endometrium, PBLs, and CL by IFNT during early pregnancy and in cell culture in response to IFNT. If RTP4 expression affects G protein-coupled receptor function, it may be important for establishment of pregnancy in domestic ruminants.

The ability of the gametes to escape detection by the immune system is vital to successful human reproduction. Furthermore, the observed capacity of the testis in some species to support tissue grafts without rejection (immunological privilege) indicates that spermatogenic cells are protected by local immunoregulatory mechanisms. One of these mechanisms involves targeting T cells for inactivation and destruction within the testicular environment. Although the fluids of the testis and ovary surrounding the developing gametes contain soluble factors that inhibit T cells, the identity of the molecule(s) responsible for this activity has been unknown. Using a specific T-cell proliferation assay to monitor bioactivity, these molecules were purified from bovine ovarian follicular fluid by methanol extraction and sequential reverse-phase HPLC (RP-HPLC). All purified active fractions coincided with the elution position on RP-HPLC of several small molecules ranging in size from 496 to 522 Da. The same molecules were localized to the immunosuppressive fractions of rat testicular interstitial fluid. The active molecules were identified, using capillary electrophoresis electrospray ionization mass spectroscopy, as lyso-glycerophosphocholines (lyso-GPCs), namely, 1-palmitoyl-sn-glycero-3-phosphocholine, 1-oleoyl-sn-glycero-3-phosphocholine, a 18:2a/lyso-GPC (putatively, 1-linoleoyl-sn-glycero-3-phosphocholine), and a 20:4a/lyso-GPC (putatively, 1-arachidonyl-sn-glycero-3-phosphocholine). Comparison of the bioactivity and mass spectroscopy profiles of two of the purified molecules with their synthetic standards confirmed the identification. These molecules inhibit T-cell proliferation in response to activation and induce apoptosis of these cells in a time- and dose-dependent manner. The emergence of gonadal lyso-GPCs as potential regulators of critical immune events opens up new avenues of inquiry into the origins of autoimmune infertility and more generally into mechanisms of peripheral immunoregulation and the development of novel immunosuppressives.

During epididymal transit, sperm acquire the ability to initiate rapid forward progressive motility on release into the female reproductive tract or physiological media. Glycolysis is the primary source of the ATP necessary for this motility in the mouse, and several novel glycolytic enzymes have been identified that are localized to the principal piece region of the flagellum. One of these is the spermatogenic cell-specific type 1 hexokinase isozyme (HK1S), the only member of the hexokinase enzyme family detected in sperm. Hexokinase activity was found to be lower in immotile sperm immediately after removal from the cauda epididymis (quiescent) than in sperm incubated in physiological medium for 5 min and showing rapid forward progressive motility (activated). However, incubating sperm in medium containing diamide, an inhibitor of disulfide bond reduction, resulted in lower motility and HK activity than in controls. HK1S was present in dimer and monomer forms in extracts of quiescent sperm but mainly as a monomer in motile sperm. A dimer-size band detected in quiescent sperm with phosphotyrosine antibody was not detected in activated sperm, and the monomer-size band was enhanced. In addition, the general protein oxido-reductase thioredoxin-1 was able to catalyze the in vitro conversion of HK1S dimers to the monomeric form. These results strongly suggest that cleavage of disulfide bonds in HK1S dimers contributes to the increases in HK activity and motility that occur when mouse sperm become activated.

The alphaT3–1 and LbetaT2 gonadotroph cell lines contain all the known factors required for expression of gonadotropin genes, yet only the LbetaT2 cells express the beta subunits. We hypothesized that comparison of their nuclear proteomes would reveal novel proteins and/or modifications that regulate expression of these genes. We identified nine proteins with different expression profiles in the two cell lines, of which several were chosen for further functional studies. Of those found at higher levels in alphaT3–1 nuclei, 1110005A23RIK was found associated with the Fshb gene promoter and repressed its expression. Transgelin 3 overexpression reduced transcript levels of Fshb, and its knockdown elevated Lhb and Cga transcript levels, indicating an ongoing repressive effect on these more highly expressed genes, possibly through altering levels of phosphorylated mitogen-activated protein kinase. Heterogeneous nuclear ribonucleoprotein A2/B1 repressed splicing of the Fshb primary transcript, which it binds in the first intron. Proteins at higher levels in LbetaT2 nuclei included prohibitin, the overexpression of which reduced promoter activity of all three gonadotropin subunits, and appeared to mediate the differential effect of GnRH on proliferation of the two cell lines; its knockdown also altered cell morphology. Two other splicing factors were also found at higher levels in LbetaT2 nuclei: the knockdown of PRPF19 or EIF4A3 decreased splicing of Lhb, or of both beta subunit transcripts, respectively. The levels of Eif4a3 mRNA were increased by activin, and both factors increased Fshb splicing. This study has revealed a number of novel factors that alter gonadotropin expression and gonadotroph function, and likely mediate or moderate effects of the regulatory hormones.

The failure to reject the semiallogenic fetus by maternal T lymphocytes suggests that potent mechanisms regulate these cells. PDCD1 is a CD28 family receptor expressed by T cells, and its ligand CD274 is strongly expressed by trophoblast cells of the human placenta. In this study, we examined whether human maternal T cells express PDCD1. Immunofluorescence examination of uterine tissues revealed PDCD1 expression on CD3 cells was low in nonpregnant endometrium but increased in first-trimester decidua and remained elevated in term decidua (P < 0.05). In addition, higher relative proportions of term decidual CD8^bright, CD4 , and regulatory T cells expressed PDCD1 in comparison to autologous peripheral blood (P < 0.05). Term decidual T cells also expressed full-length and soluble PDCD1 mRNA isoforms more abundantly than their peripheral blood counterparts (P ≤ 0.05). We also examined the effects of PDCD1:CD274 interactions in decidual T cells. Jar choriocarcinoma cells were transfected with CD274 and cocultured with activated decidual CD4 or CD8^bright T cells for 72 h followed by analysis of cytokine concentration and decidual T cell apoptosis. Compared with empty vector-transfected cells, CD274-transfected Jar cells caused a significant suppression of interferon gamma and tumor necrosis factor alpha production by CD4 (P < 0.05) but not CD8^bright T cells, while having no effect on secretion of IL10 or T cell apoptosis. These results suggest that the PDCD1:CD274 pathway functions in modification of maternal decidual lymphocyte cytokine secretion during pregnancy.

In vertebrates producing oviparous eggs, retinoids and their precursor molecules need to be deposited in oocytes during vitellogenesis. While most studies focus on the transport of retinoids and carotenoids formed outside the fish ovary and their deposition within the developing oocyte, recent investigations in mammalian species suggest the ovary is an important site for retinoid and carotenoid metabolism. Therefore, we investigated the expression of six genes (bcmo1, bcdo2, rbp1, lrat, rbp4, and stra6) associated with retinoids and carotenoids in juvenile and adult trout ovaries. Except for bcdo2, these genes were expressed in the ovary. Expression of stra6 was detected in the ovary but not in the liver. Gene expression levels of bcmo1 and stra6 were significantly higher in juvenile ovaries, in contrast to those of rbp1, rbp4, and lrat, which were similar in all tested ovarian stages. The mean values of the relative mRNA levels of the tested genes differed between the ovary and the liver. Gene transcripts of rbp4 and bcmo1 were identified by in situ hybridization in the theca layer, and all five genes were expressed in the granulosa, stromal cells, and only the early vitellogenic oocyte. The occurrence of retinol-binding protein in the theca and granulosa cells and within oocytes at all developmental stages was revealed by immunocytochemistry. These results indicate that ovarian cells express genes putatively associated with cleavage of beta-carotene, storage and mobilization of retinyl-esters, and of retinol-binding protein synthesis, suggesting a novel pathway for providing retinoids and carotenoids to developing fish ovarian follicles.

In humans, adverse pregnancy outcomes (low birth weight, prematurity, and intrauterine growth retardation) are associated with exposure to urban air pollution. Experimental data have also shown that such exposure elicits adverse reproductive outcomes. We hypothesized that the effects of urban air pollution on pregnancy outcomes could be related to changes in functional morphology of the placenta. To test this, future dams were exposed during pregestational and gestational periods to filtered or nonfiltered air in exposure chambers. Placentas were collected from near-term pregnancies and prepared for microscopical examination. Fields of view on vertical uniform random tissue slices were analyzed using stereological methods. Volumes of placental compartments were estimated, and the labyrinth was analyzed further in terms of its maternal vascular spaces, fetal capillaries, trophoblast, and exchange surface areas. From these primary data, secondary quantities were derived: vessel calibers (expressed as diameters), trophoblast thickness (arithmetic mean), and total and mass-specific morphometric diffusive conductances for oxygen of the intervascular barrier. Two-way analysis of variance showed that both periods of exposure led to significantly smaller fetal weights. Pregestational exposure to nonfiltered air led to significant increases in fetal capillary surface area and in total and mass-specific conductances. However, the calibers of maternal blood spaces were reduced. Gestational exposure to nonfiltered air was associated with reduced volumes, calibers, and surface areas of maternal blood spaces and with greater fetal capillary surfaces and diffusive conductances. The findings indicate that urban air pollution affects placental functional morphology. Fetal weights are compromised despite attempts to improve diffusive transport across the placenta.

Identifying and assessing changes in genome signaling networks will not advance the understanding of the mechanisms responsible for the temporal and spatial changes in gene expression if cells lines are not validated with respect to their phenotypes.