One of the principal objects of theoretical research in any department of knowledge is to find the point of view from which the subject appears in its greatest simplicity. (Willard Gibbs, 1881)

Omne vivum ex ovo. (Wm. Harvey, 1651)

All vertebrate follicles have the same basic structure. Viviparity also occurs in all vertebrates except birds, but it is the only form of reproduction in eutherians (“placental mammals”). Their mature follicles are vesicular, and their oocytes are yolkless. Clues to the origin of these unique characteristics are in the incidence of atresia and the role of yolk in reproduction. In broadcast spawning, atresia is as rare as it is common among eutherians and other nonspawning vertebrates. In all but the eutherians, at least the initial—and in most cases all—stages of embryogenesis depend crucially on the zygote's yolk. Eutherian reproduction, therefore, must have evolved in connection with genetic changes that caused fragility of the oocyte, instability of the follicle, and loss of the ability to produce vitellogenin (VTG), the main lipoprotein of yolk. Mutations can result in adaptations by uncovering hidden properties in a trait and/or its environment. Useful mutations in recessive alleles can spread through a population as heterozygotes, invisible until the number of homozygotes for the mutation is large enough for them to suddenly appear and form the nucleus of a new breeding population. Such a mutation probably truncated a long, oviductal-based, aplacental gestation of a small, lightly yolked zygote in an endothermic, mammal-like reptile and converted it into an early monotreme or marsupial-like mammal (pantothere). Against tremendous odds, another mutation later caused loss of the genes for VTG. The resultant yolkless zygote survived because 1) the mutation also affected a network of homeiotic genes controlling the ontogeny of the entire reproductive system and 2) the system contained enough hidden properties for the mutation to change the character of the oocyte, its granulosa cells and corpus luteum, the zygote, and the uterus in a way that virtually assured the new zygote's survival. Eutherian reproduction, however, is neither better nor worse than other forms; it is only different.

Three subfamilies of genes are acknowledged within the zona pellucida (ZP) gene family. At present, these subfamilies each have two names that are used interchangeably: ZPA or ZP2, ZPB or ZP1, and ZPC or ZP3. The ZPA genes encode the longest protein sequences and the ZPC genes the shortest. Recently, several sequences, which have no clear relationship to the three subfamilies, have been identified. These sequences include two paralogous ZP genes from Xenopus laevis and a single gene from the fish Oryzias latipes. We have conducted extensive phylogenetic analyses of the known ZP genes. As well as establishing the evolutionary relationships among these genes, the analyses make it clear that the dual nomenclature system is no longer feasible, because major paralogous groups are present in the ZPB (ZP1) family of genes of amniotes. We propose a unified system of nomenclature for the ZP gene family that removes the existing ambiguities.

Galanin is a 29-amino-acid peptide that colocalizes with GnRH in hypothalamic neurons. High concentrations of galanin are present in portal vessel blood of both male and female rats, and galanin receptors are present on gonadotropes in both sexes. Results from studies of female rats indicate that galanin acts at the level of the pituitary to directly stimulate LH secretion and also to enhance GnRH-stimulated LH secretion. The effects of galanin on pituitary LH secretion in male rats are relatively uncharacterized; thus, the present in vivo study was conducted 1) to examine the ability of galanin to affect basal or GnRH-stimulated LH secretion in male rats and 2) to determine whether the effects of galanin on LH secretion in male rats are testosterone-dependent. All three doses of galanin used (1, 5, and 10 μg/pulse) significantly enhanced GnRH-stimulated LH secretion in intact male rats. Only the highest dose of galanin directly stimulated LH secretion (without GnRH coadministration) in intact males. Galanin did not directly stimulate LH secretion or enhance GnRH-stimulated LH secretion in castrated male rats. In fact, the highest dose of galanin inhibited GnRH-stimulated LH secretion in castrated males. Upon testosterone replacement, the ability of galanin to directly stimulate LH secretion and to enhance GnRH-stimulated LH secretion was restored in castrated males. These results suggest a role for galanin in the regulation of LH release in male rats and demonstrate that testosterone upregulates the ability of the pituitary to respond to the stimulatory effects of galanin.

The aim of the present study was to determine whether the fetal lamb brain has the capacity to aromatize androgens to estrogens during the critical period for sexual differentiation. We also determined whether administration of the aromatase-inhibitor 1,4,6-androstatriene-3,17-dione (ATD) could cross the placenta and inhibit aromatase activity (AA) in fetal brain. Eight pregnant ewes were utilized. On Day 50 of pregnancy, four ewes were given ATD-filled Silastic implants, and the other four ewes received sham surgeries. The fetuses were surgically delivered 2 wk later (Day 64 of gestation). High levels of AA (0.8–1.4 pmol/h/mg protein) were present in the hypothalamus and amygdala. Lower levels (0.02–0.1 pmol/h/mg protein) were measured in brain stem regions, cortex, and olfactory bulbs. The Michaelis-Menten dissociation constant (K_m) for aromatase in the fetal sheep brain was 3–4 nM. No significant sex differences in AA were observed in brain. Treatment with ATD produced significant inhibition of AA in most brain areas but did not significantly alter serum profiles of the major sex steroids in maternal and fetal serum. Concentrations of testosterone in serum from the umbilical artery and vein were significantly greater in male than in female fetuses. No other sex differences in serum steroids were observed. These data demonstrate that high levels of AA are found in the fetal sheep hypothalamus and amygdala during the critical period for sexual differentiation. They also demonstrate that AA can be inhibited in the fetal lamb brain by treating the mother with ATD, without harming fetal development.

Our previous results showed that embryotrophic factor-3 (ETF-3) from human oviductal cells increased the size and hatching rate of mouse blastocysts in vitro. The present study investigated the production of ETF-3 by an immortalized human oviductal cell line (OE-E6/E7) and the effects of ETF-3 on the mRNA expression of mouse embryos. The ETF-3 was purified from primary oviductal cell conditioned media using sequential liquid chromatographic systems, and antiserum against ETF-3 was raised. The ETF-3-supplemented Chatot-Ziomek-Bavister medium was used to culture Day 1 MF1 × BALB/c mouse embryos for 4 days. The ETF-3 treatment significantly enhanced the mouse embryo blastulation and hatching rate. The antiserum, at concentrations of 0.03–3%, abolished the embryotrophic effect of ETF-3. Positive ETF-3 immunoreactivity was detected in the primary oviductal cells, OE-E6/E7, and blastocysts derived from ETF-3 treatment. Vero cells (African Green Monkey kidney cell line), fibroblasts, and embryos cultured in control medium did not possess ETF-3 immunoreactivity. The mRNA expression patterns of the treated embryos were studied at the blastocyst stage by mRNA differential display reverse transcription-polymerase chain reaction (DDRT-PCR). The DDRT-PCR showed that some of the mRNAs were differentially expressed after ETF-3 treatment. Twelve of the differentially expressed mRNAs that had high homology with cDNA sequences in the GenBank were selected for further characterization. The differential expression of seven of these mRNAs (ezrin, heat shock 70-kDa protein, cytochrome c oxidase subunit VIIa-L precursor, proteinase-activated receptor 2, eukaryotic translation initiation factor 2β, cullin 1, and proliferating cell nuclear antigen) was confirmed by semiquantitative RT-PCR. In conclusion, immortalized oviductal cells produce ETF-3, which influences mRNA expression of mouse blastocyst.

Mammalian oocytes are very unique cells with an unlimited developmental potential. These totipotent cells are able to remove existing gene-expression patterns and to impose new ones. However, genome reprogramming is still a mystery. Posttranslational modifications by acetylation of the N-termini portion of histones composing the nucleosome are involved in genome reprogramming. These modifications alter the higher-order chromatin structure to render the DNA accessible to the regulatory and transcriptional machinery. In the present study, we have investigated, to our knowledge for the first time, precise expression patterns of seven genes involved in chromatin structure throughout bovine embryo development. Oocytes harvested from bovine ovaries were used for in vitro production of germinal vesicle oocytes, metaphase II oocytes, 2- and 8-cell embryos, and blastocysts. Total RNA was extracted from pools (triplicates) of 20 oocytes or from embryos of each developmental stage. By means of quantitative reverse transcription-polymerase chain reaction using SYBR Green to detect double-stranded DNA, mRNA expression profiles for histone deacetylases (HDAC1, HDAC2, HDAC3, and HDAC7), histone acetyltransferases (GCN5 and HAT1), and histone H2A were established. Transcripts for all genes were detected at all stages from the oocyte to the blastocyst. The HDAC1, HDAC2 (class I HDAC), and HAT1 (type B HAT) revealed similar expression profiles. The HDAC3 (class I HDAC) tends to have an expression profile similar to those of HDAC1, HDAC2, and HAT1, whereas the HDAC7 (class II HDAC) and GCN5 (type A HAT) profiles were different from those three. These results indicate variable levels of histone deacetylases and histone acetyltransferases throughout embryonic development and may indicate the ones that are involved in somatic remodeling.

The effects of maternal 50% food restriction (FR) during the last week of gestation and/or lactation on pituitary-gonadal axis (at birth and weaning), on circulating levels of leptin (at weaning), and on the onset of puberty have been determined in rats at birth and at weaning. Maternal FR during pregnancy has no effect at term on the litter size, on the basal level of testosterone in male pups, and on the drastic surge of circulating testosterone that occurs 2 h after birth. At weaning, similar retardation of body growth is observed in male and female pups from mothers exposed to FR. This undernutrition induces the most drastic effects when it is performed during both gestation and lactation or during lactation alone. Drastic retardation of testicle growth with reduction of cross-sectional area and intratubular lumen of the seminiferous tubules is observed in male pups from mothers exposed to undernutrition during both gestation and lactation or during lactation alone. Maternal FR during the perinatal period reduces circulating levels of FSH in male pups without affecting LH and testosterone concentrations. Maternal FR does not affect circulating levels of LH, estradiol, and progesterone in female pups. Female pups from mothers exposed to FR during both gestation and lactation show a significant increase of plasma FSH as well as a drastic retardation of ovarian growth. The follicular population was also altered. The number of antral follicles of small size (vesicular follicles) was increased, although the number of antral follicles of large size (graafian follicles) was reduced. Maternal FR occurring during both late gestation and lactation (male and female pups), during lactation alone (male and female pups), or during late gestation (female pups) induces a drastic reduction of plasma leptin and fat mass in pups at weaning. The onset of puberty is delayed in pups of both sexes from mothers exposed to FR during lactation and during both gestation and lactation. In conclusion, these data demonstrate that a perinatal growth retardation induced by maternal FR has long-term consequences on both size and histology of the genitals, on plasma gonadotropins and leptin levels, on fat stores at weaning, and on the onset of puberty.

Previous reports have described that female mice deficient in cyclooxygenase-2 (COX2) are largely infertile because of failure to ovulate, poor fertilization, and defective implantation and decidualization. In the present study, we reinvestigated reproduction in these mice and found they do show a reduction in the numbers of ovulated and fertilized eggs. However, we did not observe any substantial effect on embryo implantation frequencies or an inability of COX2-deficient females to support embryo development to weaning. Pseudopregnant COX2-null recipients do not show any alteration in the timing of implantation following blastocyst transfer, but they do show a delay in the initial rate of decidual growth after implantation that lags by approximately 24 h compared to that in heterozygous or wild-type recipients. These results support previous findings that COX2 has a role in mediating the initial uterine decidual response but is not essential to sustaining decidual growth and embryo development throughout the remainder of pregnancy.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx, 20 kDa) and sperm nuclei glutathione peroxidase (snGPx, 34 kDa) are two selenoproteins present in mammalian testis and epididymal spermatozoa. They originate from the differential splicing of the PHGPx gene and appear to play important roles in sperm physiology. To determine the stages of spermatogenesis in which they are present, we compared the expression pattern of these two enzymes in highly purified populations of germ cells during specific phases of differentiation. In Northern and Western blotting experiments, both PHGPx transcript and protein were markedly expressed in pachytene spermatocytes and round spermatids. In contrast, the testis-specific snGPx was detected at both the mRNA and protein level only in haploid round spermatids. Accordingly, the developmental analysis of testicular RNAs from rats of different ages first revealed the appearance of PHGPx and snGPx transcripts at Day 20 and Day 30, respectively. Furthermore, both meiotic and postmeiotic cells contained catalytically active PHGPx/snGPx, with higher activity in the haploid cells. The intracellular distribution of PHGPx in mitochondria and nuclei of meiotic cells was demonstrated by immunocytochemical electron microscopy and Western blotting. These findings provide evidence that the PHGPx gene is differentially spliced during the meiotic prophase and haploid cell phases of spermatogenesis.

The expression and localization of GATA-4 and GATA-6 mRNAs and proteins were assessed in porcine ovaries at different stages of the estrous cycle. Reverse transcription polymerase chain reaction and Western blot analyses revealed that GATA-4 and GATA-6 transcripts and proteins were strongly expressed in granulosa cells isolated from antral follicles, intact antral follicles, corpora hemorrhagica (CH), and midluteal phase corpora lutea (CL). Immunoblot analyses showed two predominant proteins with molecular masses of approximately 53 and 55 kDa for GATA-4 and one 55-kDa protein for GATA-6. Immunohistochemical studies revealed GATA-4 and GATA-6 nuclear staining in granulosa cells of healthy primordial and primary antral follicles and antral follicle of various sizes. The percentage of immunopositive thecal cell nuclei increased with follicular development. In CH and CL, luteal cells displayed nuclear immunoreactivity for both transcription factors. Regressing CL showed a decrease in GATA-immunopositive cells. Immunoreactivity for GATA-4 and GATA-6 was present in most blood vessels. In electrophoretic mobility shift assays, nuclear protein extracts isolated from granulosa cells and CL exhibited both GATA-4 and GATA-6 binding to a GATA consensus oligonucleotide, with GATA-4 the predominant binding protein. GATA-4 and GATA-6 DNA binding was elevated in granulosa cell nuclear extracts from preovulatory (8–10 mm) follicles. Cotransfection of primary cultures of luteinizing granulosa cells with GATA-4 or GATA-6 expression vectors increased the activity of the porcine steroidogenic acute regulatory protein gene promoter significantly but did not significantly activate the inhibin α gene promoter. The detection of GATA-4 and GATA-6 mRNAs and proteins in porcine ovaries and the identification of at least one possible target gene may help to establish roles for these GATA factors in follicular development and luteal function.

The penis is unique in that it undergoes morphogenesis and differentiation primarily in the postnatal period. For complex structures such as the penis to be made from undifferentiated precursor cells, proliferation, differentiation, and patterning are required. This process involves coordinated activity of multiple signals. Sonic hedgehog (Shh) forms part of a regulatory cascade that is essential for growth and morphogenesis of many tissues. It is hypothesized that the penis utilizes regulatory mechanisms similar to those of the limb and accessory sex organs to pattern penile postnatal morphogenesis and differentiation and that the Shh cascade is critical to this process. To test this hypothesis, Shh, BMP-4, Ptc, and Hoxa-10 localization and function were examined in Sprague-Dawley rat penes by means of quantitative reverse transcription polymerase chain reaction, in situ hybridization, immunohistochemistry, and Western blotting. These genes were expressed in the penis during postnatal morphogenesis in a spatially and temporally restricted manner in adjacent layers of the corpora cavernosal sinusoids. The function of Shh and BMP-4 is to establish and maintain corpora cavernosal sinusoids. The data suggest that Ptc and Hoxa-10 are also important in penile morphogenesis. The continuing function of Shh and targets of its signaling in maintaining penile homeostasis in the adult is significant because disruption of Shh signaling affects erectile function. This is the first report that demonstrates the significant role that Shh plays in establishing and maintaining penile homeostasis and how this relates to erectile function. These studies provide valuable insight that may be applied to improve treatment options for erectile dysfunction.

The present investigation was conducted to identify and characterize an mRNA that was found by RNA differential display to be uniquely regulated at the sites of embryo implantation in mouse uterus. This mRNA was upregulated at the sites of blastocyst attachment at implantation and was identified as proprotein convertase 6 (PC6). PC6 mRNA level was low in the nonpregnant and early pregnant uterus before embryo implantation commenced (before Day 4.5, vaginal plug = Day 0). During the initiation and progression of blastocyst attachment (around Day 4.5), the mRNA was dramatically upregulated only at the implantation sites. The increased transcription was maintained on Day 5.5; the mRNA level declined slightly on Day 6.5 and then fell sharply to reach the nonpregnant level around Days 8.5–10.5. Thus, the upregulation is transient and coincides with the period of embryo attachment and implantation; it is also very specific to implantation sites. In situ hybridization analysis localized the mRNA expression predominantly in the decidual cells immediately surrounding the implanting embryo at the antimesometrial pole. Additionally, multiple mRNA species resulting from alternative splicing were observed in the uterus, as previously reported in the intestine and brain, and further analysis of these transcripts identified a uterine-specific PC6 mRNA. These data lead us to suggest that PC6 plays an important role in the processes of stromal cell decidualization and embryo implantation.

Rescue of the corpus luteum from its programmed senescence maintains progesterone production required for pregnancy. In primates, chorionic gonadotropin produced by the developing conceptus acts as the primary luteotrophic signal. The purpose of this research was to assess corpus luteum rescue by examining changes in daily urinary progesterone metabolite levels during the first week after implantation. We determined the variability in progesterone metabolite profiles and evaluated its relationship to early pregnancy loss in 120 naturally conceived human pregnancies, including 43 early pregnancy losses. In other primates, an abrupt increase in the progesterone metabolite occurs at the time of implantation. This pattern occurred in an estimated 45% of the pregnancies in the present study. In the remaining pregnancies, there was a delayed rise (18%), neither a rise or decline (22%), or a decline (15%) during the week after implantation. The estimated rate of early pregnancy loss increased across these categories (from 5% loss with an abrupt rise at implantation to 100% loss with progesterone metabolite decline). Low urinary hCG levels in early pregnancy were significant determinants of a decline in postimplantation progesterone metabolite. However, preimplantation steroid metabolite levels were not significant, suggesting no inherent problem with the corpus luteum. Examination of individual progesterone metabolite profiles in relation to hCG profiles also indicated that few losses were caused by corpus luteum failure. Delineating the functional importance of an abrupt progesterone rise at the time of implantation may provide new strategies for promoting successful implantation in assisted reproduction.

Successful implantation requires synergism between the developing embryo and the receptive endometrium. In the baboon, infusion of chorionic gonadotropin (CG) modulates both morphology and physiology of the epithelial and stromal cells of the receptive endometrium. This study explored the signal transduction pathways activated by CG in endometrial epithelial cells from baboon (BE) and human (HES). Incubations of BE and HES cells with CG did not significantly alter adenylyl cyclase activity or increase intracellular cAMP when compared with Chinese hamster ovarian cells stably transfected with the full-length human CG/luteinizing hormone (LH) receptor (CHO-LH cells). However, in BE and HES cells, CG induced the phosphorylation of several proteins, among them, extracellular signal-regulated protein kinases 1 and 2 (ERK 1/2). Phosphorylation of ERK 1/2 in uterine epithelial cells was protein kinase A (PKA) independent. This novel signaling pathway is functional because, in response to CG stimulation, prostaglandin E₂ (PGE₂) was released into the media and increased significantly 2 h following CG stimulation. CG-stimulated PGE₂ synthesis in epithelial cells was inhibited by a specific mitogen-activated protein kinase (MEK 1/2) inhibitor, PD 98059. In conclusion, immediate signal transduction pathways induced by CG in endometrial epithelial cells are cAMP independent and stimulate phosphorylation of ERK 1/2 via a MEK 1/2 pathway, leading to an increase in PGE₂ release as the possible result of cyclooxygenase-2 activation.

Use of anabolic-androgenic steroids (AASs) is becoming increasingly popular among adolescent girls, yet the effects of AASs on female physiology and development are not well understood. The present study compared the effects of chronic exposure to three individual AASs, stanozolol (0.05–5 mg/kg), 17α-methyltestosterone (0.5–5 mg/kg), and methandrostenolone (0.5–5 mg/kg) on the onset of puberty and estrous cyclicity in the rat. Female rats received daily injections of AASs for 30 days (Postnatal Day [PN] 21–51). Rats receiving the highest dose of each of the AASs (5 mg/kg) displayed vaginal opening at a younger age than rats receiving the oil vehicle. The day of first vaginal estrus was delayed in rats receiving stanozolol (5 mg/kg) or 17α-methyltestosterone (0.5–5 mg/kg) but not in rats receiving methandrostenolone. At the highest dose (5 mg/kg), each of the AASs reduced the incidence of regular estrous cyclicity during the treatment period. Concurrent administration (on PN21–51) of the androgen receptor antagonist, flutamide (10 mg/kg, twice daily), reversed the effects of 17α-methyltestosterone (5 mg/kg) on vaginal opening. Flutamide administration also eliminated the effects of stanozolol (5 mg/kg) and 17α-methyltestosterone (5 mg/kg) on the day of first vaginal estrus. In contrast, rats receiving flutamide and methandrostenolone (5 mg/kg) exhibited first vaginal estrus earlier than controls. The present results indicate that chronic exposure to AASs during development has deleterious effects on the female neuroendocrine axis and that these effects appear be mediated via multiple mechanisms.

Leptin, the 16-kDa protein product of the obese gene, was originally seen as an adipocyte-derived signaling molecule. Recently, it has been suggested to be involved in some functions during pregnancy, particularly in the placenta. In the present study, we investigated the role of leptin in the secretion of hCG, progesterone, and interleukin-6 (IL-6) by human term trophoblast cells in culture. Placentae were obtained from cesarean sections following uncomplicated pregnancies and used immediately after delivery. Leptin, hCG, progesterone, and IL-6 were measured by ELISA, RIA, and immunoradiometric assay in the cultured media of trophoblast cells cultured for 48 and 96 h. Leptin mRNA expression in these cultures was determined by reverse transcription-polymerase chain reaction. Recombinant human leptin added to primary cultures of human term placental trophoblast cells showed a stimulatory effect on hCG and IL-6 secretion and an inhibitory effect on progesterone secretion. Primary cultures of term trophoblast cells expressed leptin mRNA. All these findings suggest a role for leptin in human placental endocrine function.

Nitrergic neurotransmission triggering penile erection is mediated by nitric oxide (NO) synthesized in the cavernosal nerves of the penis by penile neuronal NO synthase (PnNOS). In the central nervous system, nNOS is activated by the N-methyl-d-aspartate receptor (NMDAR) and, presumably, is inhibited by the protein inhibitor of NOS (PIN). The PnNOS and NMDAR are expressed in the penis, and PnNOS has been localized in penile nerves. Both proteins colocalize with PIN in the hypothalamus and the spinal cord involved in the control of erection. The present study aimed to elucidate the relationship between PnNOS, PIN, and NMDAR in the penis. It was found that in the rat, PIN was expressed in the pelvic ganglion and the cavernosal nerve, and penile PIN cDNA was cloned, sequenced, and expressed. Immunohistochemistry localized PIN to the cavernosal and dorsal nerve of the penis, whereas NMDAR was not detected in the latter. Dual-fluorescence labeling showed that PnNOS colocalized with PIN in both nerves but with NMDAR only in the cavernosal nerve. Aging did not affect the mRNA levels of PnNOS, nNOS, NMDAR, and PIN. Both PIN and NMDAR were detected in penile nerves of the wild-type and nNOS^−/− mouse. The PIN protein did not inhibit or bind NOS in penile extracts, and in vivo, PIN cDNA reduced the erectile response to electrical field stimulation. In conclusion, PIN and NMDAR colocalize with PnNOS in penile nerves, but the functional significance of these protein interactions for penile erection remains to be elucidated.

Much controversy exists regarding the presence of the cadherin/catenin complex and its intracellular attachment site in the testis, which is the functional unit for actin-based cell-cell adherens junctions (AJs) in multiple epithelia. Furthermore, whether germ and Sertoli cells are equipped with the necessary AJ-associated signaling molecules to regulate this cadherin/catenin complex during spermatogenesis is not known. In the present study, it was shown that both Sertoli and germ cells indeed express N-cadherin, E-cadherin, α-catenin, β-catenin, and p120^ctn by semiquantitative reverse transcription-polymerase chain reaction and immunoblotting. Furthermore, the assembly of AJs between Sertoli and germ cells was associated with a transient induction in the steady-state mRNA and protein levels of cadherins and catenins. These analyses reveal, to our knowledge for the first time, that the testis may indeed be using the cadherin/catenin complex as one of the functional units to regulate AJ dynamics between Sertoli and germ cells in addition to α₆β₁ integrin and the nectin/afadin complex. To further confirm the existence of such a complex between Sertoli and germ cells, immunoprecipitation experiments were performed using Sertoli-germ cell lysates during AJ assembly. An anti-N-cadherin antibody can pull out β-catenin, whereas N-cadherin can also be pulled out using an anti-β-catenin antibody. To further expand and validate these in vitro biochemical studies, immunofluorescent histochemistry was performed, which colocalized N-cadherin and β-catenin to the same site of Sertoli-Sertoli and Sertoli-germ cell AJs, possibly ectoplasmic specializations near the basal compartment, at the lower third of the seminiferous epithelium in vivo as well as between Sertoli cells cultured in vitro. Furthermore, studies by cross-linking using dithiobis(succinimidylpropionate) confirmed that the cadherin/catenin complex between Sertoli cells as well as between Sertoli and germ cells indeed structurally linked to actin but not to vimentin (an intermediate filament protein) or to tubulin (a microtubule protein). These results thus unequivocally demonstrate that the cadherin/catenin complex, which can be up-regulated by testosterone, is indeed present between Sertoli and germ cells and is used for the assembly of functional AJs.

Mechanisms of antimicrobial protection in male reproductive organs are poorly understood. Defensins are antimicrobial peptides produced by many epithelial tissues. The goals of the present study were 1) to test the hypothesis that adult rat male reproductive organs express mRNA for rat β-defensin (RBD)-1 and RBD-2, 2) to examine if defensin mRNA expression in the testis and epididymis is induced by bacterial lipopolysaccharides (LPS), and 3) to investigate the effects of androgens on defensin mRNA expression in the epididymis. Total RNA from reproductive organs was analyzed by relative reverse transcription-polymerase chain reaction analysis. RBD-1 mRNA was detected in the testis. All segments of epididymis expressed equal levels of RBD-1 mRNA with higher expression than in the testis, whereas accessory sex glands showed expression equal to that in the testis. Expression of RBD-2 mRNA was primarily restricted to the penis. Effects of inflammation on defensin mRNA expression were examined in rats administered a unilateral injection of LPS from Pseudomonas aeruginosa or Escherichia coli. Expression of RBD-1 mRNA in the testis and epididymis was unaffected by LPS. To test the hypothesis that circulating androgens regulate RBD-1 mRNA expression in the epididymis, rats were subjected to bilateral orchiectomy (orch) or to orch plus a 3.5-cm implant containing testosterone. Expression of RBD-1 mRNA in the initial segment and caput was unchanged following 1-day orch but showed androgen-sensitive expression after 5 and 15 days. Expression of RBD-1 mRNA in corpus and cauda was not affected by orch. Results of this study suggest that RBD-1 may play an antimicrobial role in the testis and epididymis.

The roles of arachidonic acid (AA) and protein kinase C (PKC) during in vitro maturation-inducing hormone (MIH)-dependent meiotic resumption (maturation) and ovulation were studied in ovarian follicles of Atlantic croaker (Micropogonias undulatus). The requirement for cyclooxygenase (COX) metabolites of AA was examined using a nonspecific COX inhibitor, indomethacin (IM), as well as two COX products, prostaglandin (PG) F_2α and PGE₂, whereas the role of lipoxygenase (LOX) was investigated using a specific LOX inhibitor, nordihydroguaiaretic acid (NDGA). The involvement of PKC was examined using phorbol 12-myristate 13-acetate (PMA), a PKC activator, as well as GF109203X (GF), a specific inhibitor of PKC and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), nonspecific inhibitor of protein kinases. Genomic mechanisms were examined with the transcription-inhibitor actinomycin D (ActD) and the functionality of heterologous (oocyte-granulosa) gap junctions (GJ) with a dye transfer assay. The AA (100 μM) and PGF_2α (5 μM) did not induce maturation, and NDGA (10 μM) did not affect MIH-dependent maturation. However, IM (100 μM) partially inhibited MIH-dependent maturation. Conversely, AA and both PGs induced, and IM and NDGA inhibited, MIH-dependent ovulation in matured follicles. The PMA (1 μg/ml) did not induce maturation but caused ovulation in matured follicles, whereas PKC inhibitors (GF, 5 μM; H7, 50 μM) did not affect MIH-dependent maturation but inhibited MIH- and PMA-dependent ovulation. The PMA-dependent ovulation was inhibited by IM but not by NDGA. In addition, ActD (5 μM) blocked MIH-dependent, but not PMA-dependent, ovulation, and PGF_2α restored MIH-dependent ovulation in ActD-blocked follicles. The AA and PGs did not induce, and GF did not inhibit, MIH-dependent heterologous GJ uncoupling. In conclusion, AA and PKC mediate MIH-dependent ovulation but not meiotic resumption or heterologous GJ uncoupling in croaker follicles, but a permissive role of COX products of AA during maturation is possible. A novel model of MIH-dependent ovulation is proposed in which 1) LOX and COX metabolites of AA are both required for ovulation, but at upstream and downstream sites of the pathway, respectively, relative to PKC, and 2) PKC is downstream of genomic activation.

Follicle diameters and concentrations of follicular fluid factors were studied in the two largest follicles (F1 and F2) using F1 diameters in increments of 0.2 mm (equivalent to 4 h intervals) and extending from 7.4 to 8.4 mm (12 heifers in each of 6 groups). Changes were compared between follicles using the F2 associated with each F1-diameter group. Diameter deviation began in the 8.2-mm group as indicated by a greater (P < 0.05) diameter difference between F1 and F2 in the 8.4-mm group than in the 8.2-mm group. In the 8.0-mm group, estradiol concentrations began to increase (P < 0.05) differentially in F1 versus F2, and free insulin-like growth factor-1 (IGF-1) began to decrease differentially in F2 (P < 0.06). Combined for F1 and the associated F2, activin-A concentrations increased (P < 0.05) between the 7.6- and 8.2-mm groups and then decreased (P < 0.05). Results supported the hypothesis that estradiol and free IGF-1 concentrations simultaneously become higher in F1 than in the associated F2 by the beginning of diameter deviation. Results did not support the hypothesis that a transient elevation in activin-A is present in F1 but not in the associated F2 at the beginning of the estradiol and IGF-1 changes; instead, a mean transient elevation in activin-A occurred at this time only when data for the two follicles were combined. Comparisons between F1 and F2 also were made by independently grouping F2 and using diameter groups at 0.2-mm increments for F2 as well as for F1. In the diameter groups common to F1 and F2 (7.4, 7.6, 7.8, and 8.0 mm) there was a group effect (P < 0.003) for estradiol involving an increase (P < 0.05) beginning at the 7.6-mm group averaged over F1 and F2. For free IGF-1 concentrations, a fluctuation (a significant increase followed by a significant decrease) occurred independently in F1 between the 7.4- to 7.8-mm groups and independently in F2 between the 7.0- to 7.4-mm groups.

Tumor necrosis factor (TNF) α can induce both cell death and cell proliferation and exerts its effects by binding to either TNF receptor (TNFR) 1 or 2. When TNFα-bound TNFR2 interacts with TNFR-associated factor 2 (TRAF2), expression of survival/antiapoptotic genes is up-regulated. In the present study we determined the changes in localization of TNFα and TRAF2 and their mRNAs and the expression of TNFR2 in granulosa cells during follicular atresia in pig ovaries. In healthy follicles, intense signals for TNFα and TRAF2 and their mRNAs were demonstrated in the outer zone of the granulosa layer, where many proliferating cells and no apoptotic cells were observed. In atretic follicles, decreased or trace staining for TRAF2 and its mRNA and decreased expression of TNFR2 were observed in the granulosa layer, where many apoptotic cells were seen. These findings suggested that TNFα acts as a survival factor in granulosa cells during follicular atresia in pig ovaries.

The temporal relationship between changes in cervical dilatation, uterine electromyographic (EMG) activity, and maternal plasma concentrations of estradiol 17β (E₂), progesterone (P₄), and 13,14-dihydro-15-keto-prostaglandin-F_2α (PGFM), was investigated in six parturient cows. Calving was induced with a single injection of a synthetic analogue of prostaglandin F_2α (PG) on Day 274 of gestation. Cervical dilatation was measured continuously by measuring the transit time between two implanted ultrasound crystals while at the same time uterine EMG activity was measured through two silver electrodes sutured on the myometrial surface until the expulsive stage of calving had been reached. In blood samples collected at 4-h intervals, starting at the moment of PG injection, the mean plasma E₂ concentration gradually increased and was significantly elevated at 28 h after PG injection. At 4 h after PG treatment, the mean P₄ concentration had dropped significantly and continued to decrease until a value of around 1 ng/ml was reached, where it stayed until the onset of expulsion. Mean plasma PGFM concentrations increased steadily after PG injection, reaching significantly elevated concentrations at 20 h after treatment. In the five cows that delivered calves in anterior positions, uterine EMG activity, expressed as root mean square (RMS in μV), started to increase at a mean interval (± SD) of 13.1 ± 3.7 h following PG treatment. The increase in EMG activity was significantly correlated with changes in plasma PGFM concentrations. In these cows, dilatation of the caudal cervix started after a mean (± SD) interval of 28.5 ± 1.5 h following PG treatment and dilatation progressed at a mean (± SD) rate of 2.25 ± 0.24 cm/h. In one cow with a calf in the posterior position, uterine EMG activity and dilatation started at 15.8 h and 31.8 h, respectively, after induction of calving. We conclude that a predictable sequence of physiological changes occurs around induction of calving, which allows specific timing of future studies on cellular and biochemical changes within the cervix during parturition.

Outer dense fibers (ODFs) and the fibrous sheath (FS) are unique structures of the mammalian sperm tail. Recently, progress has been made in the molecular cloning of ODF and FS proteins, and because of this, questions addressing the morphogenesis and underlying protein network that make up sperm tail structures and their function can now be addressed. Using the N-terminal leucine zipper motif of the major ODF protein ODF1, we had previously isolated interacting proteins Odf2, Spag4, and Spag5. We report here a yeast two-hybrid strategy to isolate a novel rat testicular protein, OIP1, that binds to the evolutionarily conserved Cys-Gly-Pro repeats in the C-terminus of ODF1. OIP1 is expressed in round spermatids as well as in spermatocytes and several somatic tissues, albeit at a lower level. No expression was detectable in epididymis, heart, and smooth muscle. OIP1 protein localizes to the sperm tail in a pattern expected for an ODF1-interacting protein. OIP1 belongs to the family of RING finger proteins of the H2 subclass. Deletion of the putative RING motif significantly decreased binding to ODF1. Genomic analysis of rat Oip1 and Oip1 homologs indicates that Oip1 is highly conserved. Oip1 is subject to differential splicing and alternative polyadenylation events. It is interesting that Oip1 mRNAs have been reported that lack the exon encoding the putative RING finger.

We have obtained a PrP-Cre-ER^T transgenic mouse line (28.8) that selectively expresses in testis the tamoxifen-inducible Cre-ER^T recombinase under the control of a mouse Prion protein (PrP) promoter-containing genomic fragment. Cre-ER^T is expressed in spermatogonia and spermatocytes, but not in Sertoli and Leydig cells. We also established reporter PrP-L-EGFP-L transgenic mice harboring a LoxP-flanked enhanced green fluorescent protein (EGFP) Cre reporter cassette under the control of the same PrP promoter-containing genomic fragment that exhibits prominent EGFP expression in brain and testis. Using the PrP-L-EGFP-L as well as other Cre-reporter mice, we demonstrate that tamoxifen administration efficiently and selectively induces Cre-mediated recombination in the germ cell lineage. The established PrP-Cre-ER^T line should provide a valuable tool for studying functions of germ cell-expressed genes involved in spermatogenesis.

Sex in birds is chomosomally based (ZZ male, ZW female), but the mechanism underlying sex determination remains unknown. An unresolved question is whether Z gene dosage plays a role in avian sex determination. DMRT1 is an avian Z-linked gene that shows higher expression in male gonads during embryogenesis and has been proposed as a putative testis-determining gene in birds. The Z-linkage of this gene makes it an ideal candidate for testing the question of gene dosage in avian testis determination. A higher level of DMRT1 expression in male (ZZ) versus female (ZW) embryonic gonads may reflect the presence of two Z-linked copies in the male, or it may be due to specific and active upregulation of DMRT1 during testis formation. A functional interventionist strategy was used to distinguish between these two possibilities. DMRT1 expression was analyzed in chicken embryos during experimentally induced female-to-male sex reversal, using the aromatase enzyme inhibitor fadrozole. DMRT1 expression was analyzed by whole mount in situ hybridization and reverse transcription polymerase chain reaction (for mRNA) and indirect immunofluorescence (for protein). Female-to-male sex-reversed embryos (genetically ZW) showed elevated levels of DMRT1 expression similar to those of normal males (with two copies of the Z chromosome). Elevated levels of DMRT1 are therefore associated with testis development, both in normal males (ZZ) and in sex-reversed females (ZW). SOX9 expression was also activated during female-to-male sex reversal but appeared delayed relative to DMRT1 upregulation. These results show that testis development does not require two Z-linked copies of DMRT1, but it does involve active upregulation of the gene. Higher levels of DMRT1 expression during testis differentiation therefore do not simply reflect a gene dosage difference between the two sexes but imply active involvement in male development.

Widespread application of somatic cell cloning has been hampered by biological and technical problems, which include complicated and time-consuming procedures requiring skilled labor. Recently, zona-free techniques have been published with limited or no requirement for micromanipulators. The purpose of the present work was to optimize certain steps of the micromanipulator-free (i.e., handmade) procedure, to analyze the morphology of the developing blastocysts, and to explain factors involved in the high efficiencies observed. Optimization of the procedure included selection of the appropriate medium for enucleation, orientation of pairs at fusion, timing of fusion, and culture conditions. As a result of these improved steps, in vitro efficiency as measured by blastocysts per reconstructed embryo and blastocysts per working hour was among the highest described so far. The cattle serum used in our experiments was superior to other protein sources for in vitro embryo development. One possible explanation of this effect is the considerable mitogenic activity of the cattle serum compared with that of commercially available fetal calf serum. Morphological analysis of blastocysts by inverted microscopy, inner cell mass-trophoblast differential staining, and transmission electron microscopy revealed high average quality. A high initial pregnancy rate was achieved after the transfer of single blastocysts derived by aggregation of two nuclear transfer embryos into recipients. The improved handmade somatic cell nuclear transfer method may become a useful technology as a simple, inexpensive, and efficient alternative to traditional somatic cell nuclear transfer.

We explored a potential mechanism linking placental prostaglandins (PGs) with a fall in plasma progesterone and increased expression of uterine activation proteins in the mouse. PG endoperoxide H synthase 2 (PGHS-2) mRNA expression increased in placenta in late gestation in association with an 8-fold increase in PGF_2α concentration, reaching a peak on Gestational Day (GD) 18. This peak coincided with the final descent in plasma progesterone and birth on GD 19.3 ± 0.2. Implantation of a progesterone-releasing pellet in intact pregnant dams on GD 16 delayed birth at term until GD 20.9 ± 0.4 and inhibited the GD 18 increase in placental PGF_2α levels in conjunction with a delayed fall in plasma progesterone that reached its lowest level 1 day after term birth. The mRNA levels of uterine activation proteins, connexin-43 (CX-43), oxytocin receptor, PGF_2α receptor (FP), and PGHS-2, and the concentration of uterine PGF_2α all increased at normal term birth. At progesterone-delayed term birth on GD 19.3, even though tissue PGF_2α concentrations were at the same high levels observed at normal term birth, CX-43 and FP mRNA levels were lower than those at normal term birth, thereby possibly contributing to the delay of birth. These data are consistent with the hypotheses that fetal placental PGs affect the timing of birth by hastening luteolysis, that uterine activation initiates labor, and that birth may be delayed by blocking or decreasing the expression of two of the uterine activation proteins.

Prolactin (PRL) in fish is considered to be an osmoregulatory hormone, although some studies suggest that it may influence the production of steroid hormones in the gonads. The objective of the present study was to establish if PRL is involved in reproduction of the gilthead seabream—a protandrous hermaphrodite. Adult and juvenile gilthead seabream received implants of estradiol-17β (E₂) for 1 wk during the breeding season, and the mRNA expressions of PRL and PRL receptor (sbPRLR) were determined. Northern blot analysis revealed a single pituitary PRL transcript, the expression of which was significantly reduced by E₂ treatment in adults but significantly increased in juvenile fish. In adult gonads, four sbPRLR transcripts of 1.1, 1.3, 1.9, and 2.8 kilobases were observed. A competitive reverse transcription-polymerase chain reaction was developed and used to determine how E₂ treatment alters expression of the gonadal sbPRLR gene. Seabream PRLR was detectable in all samples analyzed by this assay. Levels of sbPRLR mRNA increased significantly (50-fold) after E₂ treatment in adults, but a 24-fold decrease was measured in juveniles. Immunohistochemistry using specific polyclonal antibodies raised against an oligopeptide from the extracellular domain of sbPRLR detected the receptor in spermatogonia and oocytes. Taken together, the preceding results suggest that in the seabream, PRL may act on both testis and ovary via its receptor and that the stage of maturity influences this process. The full characterization and relative importance of the different transcripts of sbPRLR in eliciting the action of PRL in the gonads remain to be elucidated.

Mice have been successfully cloned from both somatic cells and hybrid embryonic stem (ES) cells. Heterozygosity of the donor ES cell genome has been suggested as a crucial factor for long-term survival of cloned mice. In the present study, an inbred ES cell line, HM-1 (129/Ola), and a well-tested ES cell line, R1 (129/Sv × 129/Sv-CP), were used as donor cells to evaluate the developmental potential of nuclear transfer embryos. We found that ES cell confluence dramatically affects the developmental potential of reconstructed embryos. With the ES cell line HM-1 and 80–90% confluence, 49% of reconstructed embryos developed to the morula/blastocyst stage, 9% of these embryos developed to live pups when transferred to the surrogate mothers, and 5 of 18 live pups survived to adulthood. By contrast, at 60–70% confluence, only 22% of embryos developed to the morula/blastocyst stage, and after transfer, only a single fetus reached term. Consistent with previous reports, the nuclei of R1 ES cells were also shown to direct development to term, but no live pups were derived from cells at later passages (>20). Our results show that the developmental potential of reconstructed embryos is determined by both cell confluence and cell passage. These results also demonstrate that the inbred ES cell line, HM-1, can be used to produce viable cloned mice, although less efficiently than most heterozygous ES cell lines.

The function of mitogen-activated protein kinase (MAPK) during porcine oocyte maturation was examined by injecting oocytes with either mRNA or antisense RNA of porcine c-mos protein, an upstream kinase of MAPK. The RNAs were injected into the cytoplasm of porcine immature oocytes immediately after collection from ovaries, then the oocytes were cultured for maturation up to 48 h. The phosphorylation and activation of MAPK were observed at 6 h after injection of the c-mos mRNA injected-oocytes, whereas in control oocytes, MAPK activation was detected at 24 h of culture. The germinal vesicle breakdown (GVBD) rate at 24 h of culture was significantly higher in c-mos mRNA-injected oocytes than in control oocytes. In contrast, although injection of c-mos antisense RNA completely inhibited phosphorylation and activation of MAPK throughout the maturation period, the GVBD rate and its time course were the same in noninjected oocytes. The degree of maturation-promoting factor (MPF) activation was, however, very low in oocytes in the absence of MAPK activation. Most of those oocytes had both abnormal morphology and decondensed chromosomes at 48 h of culture. These results suggest that MAPK activation is not required for GVBD induction in porcine oocytes and that the major roles of MAPK during porcine oocyte maturation are to promote GVBD by increasing MPF activity and to arrest oocytes at the second metaphase.

Although FSH up-regulates follicular cell X-linked inhibitor of apoptosis protein (XIAP) expression and suppresses apoptosis in vivo, if these events are coincidental or causally related remains to be investigated. The present study examined the role and gonadotrophic regulation of XIAP expression during follicular development in vitro. Follicles (160–210 μm) cultured for 0–6 days with FSH (100 ng/ml) showed significant growth, as evidenced by increases in follicular size, cell number, and DNA contents. Follicular XIAP content was low in the absence of FSH but was increased by the addition of gonadotropin. Apoptosis was evident in follicles cultured without FSH but was suppressed in the presence of gonadotropin. At low FSH concentration (5 ng/ml), adenoviral XIAP sense cDNA expression increased XIAP and DNA contents, reduced apoptosis, and enhanced follicular growth. Infection of the FSH-stimulated follicles with XIAP antisense elicited opposite responses. In primary granulosa cell cultures, FSH significantly increased XIAP content, inhibited apoptosis, and decreased cell number, a response potentiated by XIAP sense expression. In conclusion, the present studies demonstrated, to our knowledge for the first time, that XIAP plays an important role in the regulation of ovarian follicular development. In addition, a follicle culture system coupled to an adenoviral gene-manipulation procedure has been established and may prove to be a useful approach in assessing the role of specific genes in follicular development and atresia.

Follicle-stimulating hormone, activin A, and transforming growth factor (TGF) α are important regulators of chicken granulosa cell (cGC) function. Hence, we aimed to test whether these growth factors are useful for establishing a suitable in vitro cell culture model system of primary cGC. Although cGC are easily isolated from distinct follicular stages, a long-term cGC culture system for in vitro studies has been unavailable. Here, we report a novel, long-term cell culture system that allows for cGC proliferation in vitro while maintaining the epithelial phenotype that granulosa cells exhibit in vivo. The cGC rapidly lose their epithelial morphology and acquire a mesenchymal or fibroblastoid phenotype when cultured in the absence of activin A. This process is strongly enhanced by TGFα, a well-known granulosa cell mitogen. However, FSH stimulates cGC proliferation without enhancing morphological changes and dedifferentiation. Interestingly, a combination of both activin A and FSH stimulates cGC proliferation and supports maintenance of differentiated epithelial morphology. Furthermore, activin A and FSH synergistically induce granulosa cell-specific differentiation markers such as inhibin α and chicken zona pellucida protein C, suggesting that cultured cGC resemble functionally differentiated granulosa cells. Our data demonstrate that activin signaling is necessary to sustain a morphologically differentiated phenotype of cGC in vitro. The results also suggest a pivotal importance of activin signaling for granulosa cell function in vivo.

Within minutes of the induction of DNA double-strand breaks in somatic cells, histone H2AX becomes phosphorylated at serine 139 and forms γ-H2AX foci at the sites of damage. These foci then play a role in recruiting DNA repair and damage-response factors and changing chromatin structure to accurately repair the damaged DNA. These γ-H2AX foci appear in response to irradiation and genotoxic stress and during V(D)J recombination and meiotic recombination. Independent of irradiation, γ-H2AX occurs in all intermediate and B spermatogonia and in preleptotene to zygotene spermatocytes. Type A spermatogonia and round spermatids do not exhibit γ-H2AX foci but show homogeneous nuclear γ-H2AX staining, whereas in pachytene spermatocytes γ-H2AX is only present in the sex vesicle. In response to ionizing radiation, γ-H2AX foci are generated in spermatogonia, spermatocytes, and round spermatids. In irradiated spermatogonia, γ-H2AX interacts with p53, which induces spermatogonial apoptosis. These events are independent of the DNA-dependent protein kinase (DNA-PK). Irradiation-independent nuclear γ-H2AX staining in leptotene spermatocytes demonstrates a function for γ-H2AX during meiosis. γ-H2AX staining in intermediate and B spermatogonia, preleptotene spermatocytes, and sex vesicles and round spermatids, however, indicates that the function of H2AX phosphorylation during spermatogenesis is not restricted to the formation of γ-H2AX foci at DNA double-strand breaks.

Wilms' tumor protein (WT1) is a transcriptional repressor essential for the development of mammalian kidneys and gonads. To gain insight into possible roles of WT1 in ovarian formation and follicular function, we studied patterns of mRNA and protein localization throughout fetal gonadal development and in ovaries of 4-wk-old and adult sheep. At Day 24 after conception, strong expression of WT1 mRNA and protein was observed in the coelomic epithelial region of the mesonephros where the gonad was forming. By Day 30, expression was observed in the surface epithelium and in many mesenchymal and endothelial cells of the gonad. Epithelial cells continued to express WT1 throughout gonadal development, as did pregranulosa cells during the process of follicular formation. However, WT1 expression was not observed in germ cells. During follicular growth, granulosa cells expressed WT1 from the type 1 (primordial) to the type 4 stages, but thereafter expression was reduced in type 5 (antral) follicles, consistent with the differentiation of granulosa cells into steroid-producing cells. The possible progenitor cells for the theca interna (i.e., the cell streams in the ovarian interstitium) expressed WT1 heterogeneously. However, differentiated theca cells in antral follicles did not express WT1. Strong expression of WT1 was observed during gonadal development, which is consistent with a role for WT1 in ovarian and follicular formation in the ewe. WT1 was identified in many cells of the neonatal and adult ovaries, including granulosa cells, suggesting that this factor is important for preantral follicular growth. However, the decline in WT1 expression in antral follicles suggests that WT1 may prevent premature differentiation of somatic cells of the follicle during early follicular growth.

Hox genes determine the formation of segmented structures during development. The epididymis shows a segmented organization in its structure and function beyond embryogenesis. This study examined the adult mouse epididymis and vas deferens for expression of 5′ hox genes and a hox-DNA binding cofactor. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed the expression of hoxa-9, hoxa-10, hoxa-11, hoxd-9, and hoxd-10 in all regions including the vas deferens. Semiquantitative RT-PCR revealed highest mRNA levels for hoxa-11 in the distal part of the epididymis and vas deferens, and this was confirmed by Northern blot analysis. To determine protein presence an antibody raised against a peptide N-terminal to the homeodomain of hoxa-11 was produced in rabbits. The antibody recognized a band of approximately 37–39 kDa in Western blot analysis. Immunohistochemistry indicated the presence of hoxa-11 in the nuclei of the epithelial cells with some staining in the cytoplasm. Staining was also detected in nuclei of interstitial cells throughout the entire organ and the vas deferens. A DNA binding cofactor for hoxa-11, Meis 1, was investigated for its presence in the epididymis. Semiquantitative RT-PCR identified both transcripts for Meis 1 (Meis 1a and Meis 1b) in all regions. Protein presence was confirmed by Western blot analysis, and this detected one band of approximately 53–55 kDa. Immunohistochemistry localized Meis 1 in the nuclei of interstitial cells throughout the entire organ and the vas deferens. Our study provides preliminary data from which we suggest the involvement of homeodomain transcription factors in the maintenance of segmental function of the adult epididymis and vas deferens.

The expression pattern and function of the murine endogenous retrovirus-like (MuERV-L) gene in mouse preimplantation embryos was investigated. MuERV-L was rapidly transcribed from the beginning of S phase (8 h after fertilization) in the first cell cycle. MuERV-L expression was completely repressed when transcription from the zygotic genome was inhibited by α-amanitin. These results reveal that MuERV-L is transcribed from the zygotic genome and that it is expressed earlier than any other genes previously reported. In addition, MuERV-L was expressed even when the first round of DNA synthesis was inhibited by aphidicolin, suggesting that its expression is controlled by the zygotic clock. The function of MuERV-L in the development of mouse embryos was also examined using antisense oligonucleotides. The developmental competence of embryos was markedly suppressed after the 4-cell stage when they were treated with antisense oligonucleotides. This result suggests that MuERV-L plays an important role in the development of mouse embryos at the early preimplantation stage.

Monoclonal antibody (mAb) MN13 labels mouse sperm head postacrosomal perinuclear theca (PT), which is possibly involved in oocyte activation during fertilization. The antigenic site is expressed after mild sonication followed by treatment with dithiothreitol (DTT) or heat (45°C), and is visible as a thick band in the postacrosomal region. The presence of protease inhibitors in the sonication medium suppresses the exposure of MN13 epitope (MN13p), suggesting the involvement of a proteolytic reaction in this process. Spermatozoa do not express MN13p after the induction of acrosome exocytosis by Ca² ionophore, zona binding, or during zona penetration, a strategy that ensures safe delivery of postacrosomal PT proteins to oocytes after fusion. MN13 labeling was not detectable during fertilization by zona-free in vitro fertilization, suggesting that the antigenic site does not react with proteolytic enzymes during sperm-oocyte fusion and the antibody does not recognize the nascent epitope. Microinjection of sperm heads prepared by sonication and DTT treatment led to the activation of metaphase II oocytes. The oocyte activating function of such sperm heads was significantly diminished after labeling with MN13 prior to intracytoplasmic sperm injection (ICSI), but labeling with antiequatorin antibody MN9 activated oocytes with a frequency similar to that of unlabeled sperm heads. The sperm heads in inactive oocytes formed premature chromosome condensations (PCCs), which were invested by independent metaphase-like spindles. These observations indicate that the postacrosomal PT recognized by mAb MN13 is involved in oocyte activation. MN13p is dissociated from sperm heads during the early stages of decondensation after ICSI. In activated oocytes, MN13-labeled fine granules were redistributed in the midzone spindle region, whereas in inactive oocytes they formed a ring around the polar regions of the metaphase II and PCC spindles.

Changes in binding affinity, acrosomal status, and motility of living sperm on the zona pellucida were for the first time in any mammalian species directly observed and analyzed with video microscopy. A single zona was air-dried and rehydrated on a microscope slide, and a coverslip supported by glass beads was added. Capacitated sperm were added together with Alexa-SBTI, a probe for acrosin that can detect the acrosome reaction. The heads of loosely attached sperm oscillated on the zona and the flagella beat symmetrically with a sigmoid-shaped waveform. Tight binding was observed after 16 sec as the sperm head became fixed in place on the zona. The shape of the flagellar beat simultaneously shifted to a more rigid, C-shaped waveform. The first signs of the acrosome reaction were detected within 11 sec of tight binding. Rapid flushing removed approximately 65% of sperm that were loosely attached but only 2% of those that were tightly bound. In the 2 min following the onset of tight binding, the lateral displacement of the flagellum increased by approximately 30% and the beat frequency decreased by 25%. Lignosulfonic acid (LSA) inhibited loose sperm attachment and the development of tight binding. LSA had no effect on the time of the acrosome reaction following tight binding or on changes in motility that followed tight binding. These data suggest that LSA affects the initial attachment or docking of sperm to the zona, a step that may align or recruit one or more specific zona receptors to be responsible for mediating the acrosome reaction.

Interferon-tau (IFN-τ) is produced by the trophoblast prior to implantation in ruminants. It is involved in maternal recognition of pregnancy, and is a pleiotropic molecule that can alter the synthesis of endometrial proteins and inhibit proliferation of some cells. We have observed that IFN-τ reduces the DNA content in cultures of bovine endometrial epithelial cells; therefore, the objective of this study was to determine whether IFN-τ would induce apoptosis in bovine endometrial cells. Epithelial cells were prepared, cultured to confluence, and then incubated for 24 or 48 h in the presence or absence of 10 ng/ml progesterone, 100 ng/ml IFN-τ, or 10 μg/ml cycloheximide (CHX; an apoptosis inducer used as a positive control). Cells undergoing apoptosis exhibit such characteristics as the appearance of apoptotic bodies and DNA fragmentation. The incidence of apoptosis was assessed by using TUNEL, DNA fragmentation analysis, and Western blot analysis of Bax-α protein expression. The results showed that IFN-τ and CHX significantly increased the percentage of cells with apoptotic nuclei (33.6% and 44.8%, respectively) compared with controls (11.7%; P < 0.05). Progesterone treatment of the cells significantly inhibited the ability of IFN-τ to induce apoptosis (14.6%) compared with IFN-τ alone (33.6%; P < 0.05). DNA fragmentation analysis showed that INF-τ and CHX treatment resulted in an increase in the appearance of DNA laddering compared with that in untreated control cultures. Western blot analysis showed that IFN-τ and CHX treatment resulted in a greater expression of the proapoptotic protein Bax-α compared with that in control cultures. These data demonstrate that IFN-τ can induce apoptosis in bovine uterine epithelial cells and that this effect is modulated by progesterone. We speculate that IFN-τ might play a critical role in the remodeling of the endometrium around the time of implantation.

The finding of large, stage-specific changes in secretion of procathepsin L by rat Sertoli cells has led to the hypothesis that this proenzyme promotes the survival, replication, or differentiation of spermatogenic cells. Experiments described herein used a mouse model to test this hypothesis. To prove that mice are appropriate for this purpose, we first demonstrate that mature mouse Sertoli cells express cathepsin L mRNA in the same stage-specific manner as rat Sertoli cells and they also secrete procathepsin L. To test whether catalytically active cathepsin L is required for normal spermatogenesis, we examined the testes of 110- to 120-day-old furless mice, which express catalytically inactive cathepsin L. Morphologic examination of testes of furless mice revealed both normal and atrophic seminiferous tubules. Enumeration of atrophic tubules in furless and control mice demonstrates that lack of functional cathepsin L results in a 12-fold increase in seminiferous tubule atrophy. To determine whether lack of functional cathepsin L affects the production of male germ cells in apparently normal, nonatrophic tubules, we compared numbers in control and furless mice of preleptotene spermatocytes, pachytene spermatocytes, and round spermatids per Sertoli cell. Results demonstrate that the lack of functional cathepsin L causes a 16% reduction in formation of preleptotene spermatocytes and a 25% reduction in differentiation of these cells into pachytene spermatocyte. These results suggest that procathepsin L either directly or indirectly has two distinct functions in the testis. This proenzyme prevents atrophy of seminiferous tubules and promotes the formation of preleptotene spermatocytes and the differentiation of these meiotic cells into pachytene spermatocytes.

Antibodies against ubiquitin, a universal proteolytic marker, show increased cross-reactivity with defective spermatozoa in men and bulls. We investigated sperm ubiquitination in the stallion, a seasonally polyestrous mammal. Immunofluorescence and immunoelectron microscopy demonstrated that anti-ubiquitin antibodies bind to the surface of both membrane-intact and aldehyde-fixed spermatozoa. Cross-reactivity to the ubiquitin-conjugating enzyme E2 was also detected in sperm. Immunohistochemistry showed that ubiquitinated spermatozoa were first detected in the caput epididymis, coincident with a strong accumulation of ubiquitin and ubiquitin C-terminal hydrolase, protein gene product 9.5, in the apical stereocilia of the epididymal epithelium. Testicular spermatozoa did not display significant ubiquitin cross-reactivity. Similarly, lesser accumulation of ubiquitin cross-reactive substrates was identified in the accessory sex glands. Semen samples were collected from three fertile stallions and one subfertile stallion between December and February and probed for ubiquitin by flow cytometry and immunoblotting. Flow cytometric analysis showed that sperm from the subfertile stallion had higher ubiquitin levels than sperm from the other three stallions. In addition, immunoblot analysis of sperm proteins from the subfertile stallion showed two unique ubiquitin cross-reactive bands that were not present in sperm extracts from the three fertile stallions. To screen for a possible role for ubiquitin in seasonal changes in sperm production, semen samples from two fertile stallions were collected in March, June, September, and December and subjected to a flow cytometric ubiquitin assay. The lowest levels of ubiquitin-labeled sperm were found in March, approximately coincident with the onset of the natural horse breeding season. A progressive increase in sperm ubiquitin levels was found during summer and fall, with a peak in December. These data suggest that stallion sperm are differentially ubiquitinated during epididymal maturation and that this ubiquitination may reflect changes in sperm numbers and semen quality. The association between changes in sperm ubiquitination and seasonal changes in sperm production will be subjected to further studies in a larger cohort of animals.

Mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) is regulated by multiple promoters in a tissue-specific manner. We characterized the testis-specific promoter C of the mGPDH gene and investigated the cellular localization of mGPDH within the testis. Electrophoretic mobility shift experiments identified a cAMP-response element (CRE) site at −57 that was active in the testis. An in vitro-translated CRE modulator (CREM) protein was able to bind this CRE site, and an anti-CREM antibody interfered with this complex. Ectopic expression of the testis-specific transcriptional activator CREMτ and protein kinase A in human hepatocarcinoma HepG2 cells activated a promoter C-driven luciferase construct in transient transfection experiments. Furthermore, mGPDH expression was undetectable in testis of CREM-deficient mice. The cellular localization of mGPDH expression and translation in adult rat testis was determined by in situ hybridization and immunohistochemistry techniques. The mGPDH transcripts were detected solely in postmeiotic germ cells. Expression of mGPDH was restricted from round spermatids to early elongating spermatids. The mGPDH protein was delayed in postmeiotic germ cells, restricted from late elongating spermatids to mature spermatids. Our results indicate that rat mGPDH is expressed by a testis-specific promoter from haploid male germ cells in a stage-specific manner.