EDITOR'S PREFACE: The first paper in this issue of Biology of Reproduction is a minireview entitled “Getting Sperm and Egg Together: Things Conserved and Things Diverged,” by Janice P. Evans. This minireview is derived from talks delivered as part of the BioPore Minisymposium: The Egg Surface at the 32nd annual meeting of the Society for the Study of Reproduction, 31 July–3 August 1999 in Pullman, Washington. The minisymposium, organized by Victor D. Vacquier, included the following presentations: “An Hypothesis to Explain the Evolution of Species-Specific Fertilization” by Victor D. Vacquier, “Molecular Biology of the Zona Pellucida: Genetic Mutations and Fertility” by Jurrein Dean, and “Sperm-Egg Membrane Interactions During Fertilizations” by Janice P. Evans. This minireview was submitted to the Editorial Office of Biology of Reproduction and then subjected to same standards of peer review as all manuscripts published in the journal.

Virendra B. Mahesh, Ph.D., D.Phil., Editor-in-Chief, Biology of Reproduction

Sperm-egg interactions occur at multiple levels on the egg surface, first with the egg's extracellular matrix and then with the egg's plasma membrane. The BioPore minisymposium on “The Egg Surface” at the 1999 annual meeting of the Society for the Study of Reproduction highlighted a series of events underlying successful interactions of the sperm with the egg: 1) composition, synthesis, and assembly of the mouse egg's extracellular matrix, the zona pellucida, during oogenesis; 2) oocyte maturation and development of the sperm-binding domain of mouse eggs; and 3) characterization of functional domains in different sperm ligands (fertilin-α and fertilin-β in the mouse and lysin in the abalone) that recognize cognate binding sites on the egg surface. Data that were presented are reviewed here and discussed with respect to conserved and divergent features of gamete functions.

Nuclear receptor coactivators associate in a ligand-dependent manner with estrogen receptors (ER) and other nuclear receptors, and they enhance ligand-dependent transcriptional activation. This study examined basal coactivator expression in rat uterus to investigate if expression of these genes is regulated by estradiol-17β or tamoxifen. Ovariectomized mature and immature rats were injected with estradiol-17β, tamoxifen, or vehicle (i.e., sesame oil) alone. Uteri were collected and analyzed for changes in coactivator mRNA expression using Northern blot and in situ hybridization analyses. Constitutive uterine mRNA expression of switch protein for antagonist (SPA), SRC-1, GRIP1, RAC3, RIP140, and p300 mRNAs was observed in control uteri, and treatment with ER ligands did not alter coactivator mRNA levels. The data suggest that expression of these coactivator genes is not sensitive to estradiol or tamoxifen in the rat uterus. No cell type-specific pattern of expression was apparent in uterine sections from mature and immature rats; however, silver grains were more abundant in luminal and glandular epithelial cells compared with the stroma and myometrium, indicating that coactivator mRNA levels vary among the uterine compartments. Thus, to our knowledge, we show for the first time that there is constitutive expression of several uterine nuclear receptor coactivators in a physiological setting that remains insensitive to estrogenic regulation. Furthermore, we speculate that higher constitutive levels of coactivator expression in glandular and luminal epithelial cells may be associated with increased hormonal responsiveness by these uterine compartments.

Androgen is essential for maintenance of spermatogenesis in the testis and for maturation of spermatozoa in the epididymis. The effects of androgen are mediated through its receptor (AR), the levels of which are, in turn, regulated by androgen. Previous studies have shown that AR concentrations in Leydig and Sertoli cells are differentially regulated during development. The aim of the present study was to determine if cell-type-specific regulation of AR by androgen occurs in testicular and epididymal cells during adulthood. Adult male rats were treated with the LHRH-antagonist Azaline B (100 g/day) by osmotic pump for 1, 2, 3, 4, or 8 wk to suppress endogenous androgen, with identical numbers of intact control animals at each time period. An androgen replacement group was simultaneously treated with the antagonist and a synthetic androgen, 7α-methyl-19-nortestosterone (MENT), during the final 4 wk of the experiment. Levels of nuclear AR protein in specific cell types were quantified by immunohistochemistry in conjunction with computer-assisted image analysis. Levels of AR in testicular cells declined sharply after treatment with the LHRH antagonist. In Sertoli cells, nuclear AR levels decreased to 8% of control (P < 0.01) after 4 wk treatment; and to 12% and 17% of control (P < 0.01) in Leydig and myoid cells, respectively. Androgen replacement resulted in complete recovery of nuclear AR levels in Sertoli cells (93%, P > 0.05) but in only partial recovery in myoid (69%, P < 0.01) and Leydig cells (56%, P < 0.01). In the epididymis, tubular epithelial cells and stromal cells differed in their responses to the LHRH antagonist. After 1 wk, nuclear AR levels in caput stromal cells decreased dramatically to 34% of control (P < 0.01) and in cauda stromal cells to 43% (P < 0.01). In contrast, the decline of AR levels in epididymal epithelial cells was not as dramatic as that in stromal cells. After 1 wk, the decline in the caput and cauda was to 87% and 76% of control, respectively. After 8 wk, nuclear AR levels in stromal cells further declined to 1.1% in caput and 1.4% in cauda, whereas in the epithelial cells, a smaller decline in nuclear AR was noted (to 30% in the caput and 45% in the cauda). After androgen replacement with MENT, nuclear AR levels recovered to more than 90% of control in both epididymal cell types. These results indicate that AR levels in the nuclei of adult Sertoli cells depend mainly on the level of androgen, whereas in the adult Leydig and myoid cells, the androgen dependency is more limited. The results also indicate that in the epididymis, stromal cells are more sensitive than epithelial cells to the regulation of AR levels by androgen.

Luteal regression is initiated by prostaglandin F_2α (PGF_2α). In domestic species and primates, demise of the corpus luteum (CL) enables development of a new preovulatory follicle. However, during early stages of the cycle, which are characterized by massive neovascularization, the CL is refractory to PGF_2α. Our previous studies showed that endothelin-1 (ET-1), which is produced by the endothelial cells lining these blood vessels, plays a crucial role during PGF_2α-induced luteolysis. Therefore, in this study, we compared the effects of PGF_2α administered at the early and mid luteal phases on ET-1 and its type A receptors (ETA-R) along with plasma ET-1 and progesterone concentrations, and the mRNA levels of PGF_2α receptors (PGF_2α-R) and steroidogenic genes. As expected, ET-1 and ETA-R mRNA levels were markedly induced in midcycle CL exposed to luteolytic dose of PGF_2α analogue (Cloprostenol). In contrast, neither ET-1 mRNA nor its receptors were elevated when the same dose of PGF_2α analogue was administered on Day 4 of the cycle. In accordance with ET-1 expression within the CL, plasma ET-1 concentrations were significantly elevated 24 h after PGF_2α injection only on Day 10 of the cycle. The steroidogenic capacity of the CL (plasma progesterone as well as the mRNA levels of steroidogenic acute regulatory protein and cytochrome P450_scc) was only affected when PGF_2α was administered during midcycle. Nevertheless, PGF_2α elicited certain responses in the early CL: progesterone and oxytocin secretion were elevated, and PGF_2α-R was transiently affected. Such effects probably result from PGF_2α acting on luteal steroidogenic cells. These findings may suggest, however, that the cell type mediating the luteolytic actions of PGF_2α, possibly the endothelium, could yet be nonresponsive during the early luteal phase.

Involvement of estradiol in the deviation in growth rates between the two largest follicles of a wave was studied in 39 heifers. In experiment 1, the largest follicle remained intact in a control group and was ablated in five estradiol-treated groups when the largest follicle reached 8.5 mm or larger (expected beginning of deviation; Hour 0). The ablation groups were given a single injection of 0, 0.004, 0.02, 0.1, or 0.5 mg of estradiol. Blood samples were taken from a jugular vein every hour at Hours 0 to 16. By Hour 8, FSH concentrations were greater (P < 0.05) in the ablation group that received 0 mg of estradiol than in the controls. Among the estradiol groups, that receiving 0.02 mg had the lowest detectable increase in estradiol. In this group, FSH concentrations were not suppressed below the control concentrations, but the increase in FSH concentrations following ablation of the largest follicle was delayed for 2 or 3 h. This delay in the increase of FSH concentrations corresponded to the hours that estradiol was maximal. In experiment 2, blood samples were taken every 4 h from the caudal vena cava cranial to the junction with the ovarian veins in heifers with the largest follicle intact (controls) or ablated at 8.5 mm or larger (Hour 0). Averaged over Hours 4 to 48, estradiol concentrations were higher (P < 0.04) in the controls than in the ablation group. During Hours 0 to 12, estradiol concentrations increased (P < 0.05) in the controls, whereas FSH concentrations decreased (P < 0.05). In the ablation group, estradiol concentrations were lower than in the controls by Hour 4, and FSH concentrations increased (P < 0.05) between Hours 4 and 12. These results support the hypothesis that the largest follicle releases increased estradiol into the blood at the beginning of follicular deviation, and that the released estradiol is involved in the continuing depression of FSH concentrations to below the requirement of the smaller follicles.

We recently showed that insulin-like growth factor-binding protein-4 (IGFBP-4) proteolytic degradation in ovine preovulatory ovarian follicles is IGF-dependent and regulated by the heparin-binding domain (HBD) from IGFBP-3 and from connective tissue growth factor (CTGF), heparan/heparin-interacting protein (HIP), and vitronectin. The present study investigated regulation of IGFBP-4 proteolytic degradation in porcine, bovine, and equine ovarian preovulatory follicles. Follicular fluid from such preovulatory follicles contains proteolytic activity, degrading exogenous IGFBP-4. An excess of IGF-I enhanced IGFBP-4 degradation. In contrast, IGFBP-2 or -3 or monoclonal antibodies against IGF-I or -II dose-dependently inhibited IGFBP-4 degradation, and IGF-I or -II reversed this inhibition in a dose-dependent manner. Heparin-binding peptides derived from the C-terminal domain of IGFBP-3 or -5 inhibited IGFBP-4 degradation. Other heparin-binding peptides derived from CTGF, HIP, and vitronectin also inhibited IGFBP-4 degradation, except in porcine follicles. Finally, IGFBP-3 that was mutated in its HBD was less effective at inhibiting IGFBP-4 degradation. Thus, in bovine, porcine, and equine preovulatory follicles, IGFBP-4 proteolytic degradation both depends on IGFs and is inhibited by peptides containing HBD. Overall, these results suggest that during terminal development of follicles to the preovulatory stage in domestic animal species, the increase in IGF bioavailability might enhance IGFBP-4 degradation. In contrast, in atretic follicles, the decrease in IGF bioavailability, resulting partly from the increase in IGFBP-2 (sow, heifer, mare) and IGFBP-5 (heifer) expression would participate in the decrease of IGFBP-4 degradation. In bovine atretic follicles, IGFBP-5 would also strengthen the inhibition of IGFBP-4 degradation by direct interaction of its HBD with the protease. The involvement of other HBD-containing proteins in the modulation of intrafollicular proteases degrading IGFBP-4 remains to be investigated.

Progress in research on initiation of folliculogenesis has progressed slowly because of a lack of markers for early folliculogenesis. The rabbit zona pellucida protein (ZP1) is synthesized in follicles during early stages of folliculogenesis. In order to establish ZP1 as a marker for initiation of folliculogenesis, in situ hybridization was used to localize ZP1 mRNA in immature follicles. ZP1 mRNA was first detected in oocytes of some but not all primordial follicles. The primordial follicles expressing ZP1 mRNA were located at the cortico-medullary junction, indicating that they were newly activated follicles. ZP1 mRNA accumulated in oocytes of intermediate, primary, and secondary follicles. In contrast, ZP1 mRNA was first detectable in granulosa cells of intermediate follicles and is present in cuboidal granulosa cells of primary and early secondary follicles, but was undetectable in granulosa cells of more mature follicles. These data demonstrate that 1) ZP1 mRNA is expressed in both oocytes and granulosa cells, 2) ZP1 mRNA is initially expressed in oocytes of activated follicles, and 3) ZP1 mRNA is transiently expressed in granulosa cells during early stages of folliculogenesis. Therefore, rabbit ZP1 is a molecular marker that can be used in future studies to measure initiation of folliculogenesis.

Expression of the diphtheria toxin A-chain gene was directed to the male germ line by fusion to 1 kilobase of the 5′-flanking DNA of the rat histone H1t gene. Two independent lines of mice were established that expressed the toxic transgene. Female carriers were fertile; males were sterile although otherwise apparently normal. Adult transgenic males had very small testes that were virtually devoid of germ cells. A developmental study showed that germ cells survived until late fetal life but that testes of 3-day-old transgenic mice were severely depleted of prospermatogonia. During postnatal development of transgenic animals, remaining germ cells progressed to the pachytene stage of meiosis in 10% to 30% of tubular cross sections but degenerated before the completion of meiosis. By 3 mo of age the residual germ cells had almost completely disappeared. These transgenic lines demonstrate the complete tissue specificity of the H1t promoter and reveal a period of its activity just prior to formation of the definitive adult spermatogonial stem cell population. Whereas full expression of H1t occurs only in mid to late pachytene spermatocytes, one or more of the factors that impart tissue specificity to its expression must be transiently activated in the neonatal germ line. This report discusses the possibility that this genetic technique for eliminating germ cells may have practical application in making recipients for spermatogonial stem cell transplantation.

Antiluteolytic actions of bovine interferon-tau (bIFN-τ) require suppression of prostaglandin F_2α (PGF_2α) production. Our objective was to test whether bIFN-τ could block PGF_2α production and synthesis of phospholipase A₂ (PLA₂) and cyclooxygenase-2 (COX-2) enzymes induced by a protein kinase C (PKC) stimulator (phorbol 12,13 dibutyrate; PDBu). Bovine endometrial epithelial (BEND) cells were treated with PDBu in the presence or absence of bIFN-τ. Medium samples were analyzed for concentrations of PGF_2α, whole-cell extracts were analyzed for abundance of PLA₂ and COX-2 by immunoblotting, and RNA extracts were examined for steady-state levels of COX-2 mRNA by Northern blotting. The PDBu stimulated production of PGF_2α between 3 and 12 h, levels of COX-2 mRNA by 3 h and protein expression of COX-2 and PLA₂ by 6 and 12 h, respectively. Added concomitantly with PDBu, bIFN-τ suppressed PGF_2α production, steady-state levels of COX-2 mRNA, and expression of COX-2 and PLA₂ proteins. Added after a 3-h stimulation with PDBu alone, bIFN-τ suppressed PGF_2α production after 1 h. Bovine IFN-τ inhibited intracellular mechanisms responsible for PGF_2α production in BEND cells, and this could be through both cytosolic and nuclear actions.

There is recent evidence that mouse and human spermatozoa contain constitutive nitric oxide synthase (cNOS) and can synthesize nitric oxide. The aim of this study was to investigate whether the inhibition of human sperm cNOS could affect sperm-oocyte fusion and sperm binding to the zona pellucida (ZP). N^G-nitro-l-arginine methyl ester (l-NAME) was used as cNOS inhibitor. Sperm-oocyte fusion was evaluated using the hamster egg penetration test (HEPT). The ZP binding was evaluated using the hemizona assay. l-NAME added from the onset of capacitation strongly inhibited sperm-oocyte fusion. This inhibitory effect was dose dependent, stereospecific, and suppressed by l-arginine in a dose-dependent manner. l-NAME also inhibited sperm-oocyte fusion in the HEPT enhanced with progesterone (P), where P (5 μM) was added for 15 min to capacitated sperm. A lesser but significant inhibition was also observed when sperm suspensions were exposed to l-NAME following capacitation in both versions of HEPT. On the contrary, l-NAME did not affect ZP binding. In conclusion, the present study provides the evidence that cNOS plays a role in the human sperm's capacity to fuse with oocyte but not in the ZP binding.

The implanting blastocyst must appose and adhere to the endometrial epithelium and, subsequently, invade it. Locally regulated uterine epithelial apoptosis induced by the embryo is a crucial step of the epithelial invasion in rodents. To address the physiological relevance of this process in humans, we investigated the effect of single human blastocysts on the regulation of apoptosis in cultured human endometrial epithelial cells (hEEC) in both apposition and adhesion phases of implantation. Here, we report a co-ordinated embryonic regulation of hEEC apoptosis. In the apposition phase, the presence of a blastocyst rescues hEEC from the apoptotic pathway. However, when the human blastocyst adheres to the hEEC monolayer, it induces a paracrine apoptotic reaction. Fas ligand (Fas-L) was present at the embryonic trophoectoderm. Fas was localized at the apical cell surface of hEEC, and flow cytometry revealed that 60% of hEEC express Fas. Neutralizing adhesion assays revealed that the Fas/Fas-L death system may be an important mechanism to cross the epithelial barrier, which is crucial for embryonic adhesion, and the manipulation of this system could have potential clinical implications as an interceptive mechanism.

Testicular recrudescence in male black bears (Ursus americanus) is initiated in January and completed in May. The goals of this study in the black bear were to determine 1) if testicular abundance of LH-receptor (LHr), FSH-receptor (FSHr), and prolactin-receptor (PRLr) mRNA changes during recrudescence; 2) if these changes in mRNA abundance are associated with changes in serum LH, PRL, and testosterone (T) concentrations; and 3) if the spring increase in serum PRL concentrations is required for testicular recrudescence. Serum was obtained monthly from nine male bears for 2 yr, except in July and August. To suppress endogenous PRL, four bears were treated with Parlodel LAR, 50 mg per 70 kg body weight, monthly from January through May, whereas five bears served as controls. Testicular biopsies were obtained in January, March, and May and analyzed for LHr, FSHr, and PRLr mRNA abundance using reverse transcriptase-competitive polymerase chain reaction. The LHr and PRLr mRNA abundance was low in January, increased in March, and remained high in May, whereas the FSHr mRNA abundance remained constant. Serum concentrations of PRL and T increased in March, coincident with the increase in testicular LHr and PRLr mRNA abundance. Suppression of serum PRL concentrations during testicular recrudescence 1) prevented the increase in testicular LHr and PRLr mRNA abundance observed among control bears in March, 2) lowered serum T concentrations in March and April, and 3) resulted in reduced testis size in May. We conclude that testicular LHr and PRLr mRNA are seasonally regulated, and that PRL has a role in testicular recrudescence in the black bear.

Contractions of seminiferous tubules and epididymal duct walls promote spermiation and sperm transfer, and they are thought to be stimulated by the related peptides oxytocin and vasopressin. This study tested the hypothesis that if oxytocin and/or vasopressin play a physiological role in sperm shedding and transport, then local or circulating concentrations of these peptides would increase during puberty. Testes, epididymides, and trunk blood of sheep at stages during the first spermatogenic wave were collected, and radioimmunoassay measured significant increases in testicular and epididymal oxytocin during spermatogenesis. No changes were measured in circulating oxytocin or in local or circulating vasopressin. Localization and synthesis was investigated by immunohistochemistry and Western blot analysis employing antibodies recognizing epitopes of either oxytocin, oxytocin-associated neurophysin, vasopressin, or vasopressin-associated neurophysin. Marked expression of both oxytocin and its associated neurophysin in testicular Leydig and epididymal principal cells was seen, and weak neurophysin immunoreactivity was also identified in Sertoli cells. The intercellular distribution of oxytocin varied between regions of the epididymis, suggesting several roles for oxytocin. Vasopressin synthesis was not apparent in either tissue. These results confirm the presence and development of paracrine oxytocinergic systems in the ram testis and epididymis of ram during puberty while questioning the physiological importance of vasopressin.

The precise mechanism for the initiation of follicle growth and progression through the earliest stages of follicle development remains largely unknown. Activins play a role during early follicle development, and evidence suggests that the extracellular matrix plays a role during later stages of follicular growth. We investigated the role of activin-A and extracellular matrix in follicle growth initiation and early follicular development in the mouse ovary. Ovaries were collected from 5-day-old mice and cultured for 10 days on polylysine, collagen, or laminin in the presence or absence of recombinant human activin-A. Follicle density, indices of follicle growth initiation (primary:primordial follicle [PY:PD] and primary:total follicle [PY:TF] ratios), ratios of multilayer follicle:total follicle (ML:TF), and follicle growth rates were compared between groups. Follicle densities were significantly higher in the extracellular matrix treatment group compared with the polylysine group (P < 0.01). Also, compared with polylysine, both collagen and laminin significantly increased indices of follicle growth initiation (PY:PD ratio: P < 0.001, odds ratio of 3.3; PY:TF ratio: P < 0.001, odds ratio of 2.5), and these were not altered by activin treatment. In the absence of activin-A, exposure to neither collagen nor laminin had an effect on multilayer follicle development. When activin-A was added, collagen and laminin had opposing effects on multilayer follicle development. Activin-A stimulated multilayer follicle development in the presence of laminin (ML:TF ratio: P = 0.01, odds ratio of 10.8), whereas it suppressed follicle growth in collagen (P = 0.01). Activin-A did not affect the ML:TF ratio in the polylysine-treated groups. These results strongly suggest that extracellular matrix components and activin-A interact with each other, and that they regulate follicle growth initiation and multilayer follicle development.

Rat epididymal protein DE associates with the sperm surface during epididymal maturation and is a candidate molecule for mediating gamete membrane fusion in the rat. Here, we provide evidence supporting a role for DE in mouse sperm-egg fusion. Western blot studies indicated that the antibody against rat protein DE can recognize the mouse homologue in both epididymal tissue and sperm extracts. Indirect immunofluorescence studies using this antibody localized the protein on the dorsal region of the acrosome. Experiments in which zona-free mouse eggs were coincubated with mouse capacitated sperm in the presence of DE showed a significant and concentration-dependent inhibition in the percentage of penetrated eggs, with no effect on either the percentage of oocytes with bound sperm or the number of sperm bound per egg. Immunofluorescence experiments revealed specific DE-binding sites on the fusogenic region of mouse eggs. Because mouse sperm can penetrate zona-free rat eggs, the participation of DE in this interaction was also investigated. The presence of the protein during gamete coincubation produced a significant reduction in the percentage of penetrated eggs, without affecting the binding of sperm to the oolemma. These observations support the involvement of DE in an event subsequent to sperm-egg binding and leading to fusion in both homologous (mouse-mouse) and heterologous (mouse-rat) sperm-egg interaction. The lack of disintegrin domains in DE indicates that the protein interacts with its egg-binding sites through a novel mechanism that does not involve the reported disintegrin-integrin interaction.

Formation of mature spermatozoa involves a series of dramatic molecular and morphological changes in the male germ cell lineage. These changes result from the temporally regulated transcription and translation of several testis-specific gene products. Here, we describe a novel, testis-specific protein designated SPAN-X for sperm protein associated with the nucleus on the X chromosome. SPAN-X sequences showed no significant similarity with known cDNA or peptide sequences. The SPAN-X peptide sequences contained three overlapping consensus nuclear localization signals, a high percentage (33%–37%) of charged amino acid residues, and a relatively acidic isoelectric point (pI; 4.88–6.05). Northern analysis of mRNA from multiple human tissues identified a SPAN-X transcript exclusively in the testis. In situ hybridization of human testes sections showed SPAN-X mRNA expression in haploid, round, and elongating spermatids. The SPANX gene was mapped to chromosome Xq27.1 by fluorescence in situ hybridization and by Southern blot analysis of human/mouse somatic cell hybrids. On Western blots of human sperm proteins, antirecombinant SPAN-X antibodies reacted with broad bands migrating between 15–20 kDa. Immunofluorescent labeling of human spermatozoa demonstrated SPAN-X localization to nuclear craters and cytoplasmic droplets. Expression of SPAN-X, an X-linked gene product, exclusively in haploid spermatids leads to interesting questions regarding the transcription of sex-linked genes during spermiogenesis.

A rapid in vitro method has been developed to detect early effects of cytostatic drugs on rat spermatogenesis. The induction of programmed cell death (apoptosis) and changes in DNA synthesis induced by doxorubicin and idarubicin were measured in specific stages of the cycle of seminiferous epithelium including mitotic (stage V) and meiotic (stage VIII-IX) S-phase cells. The model was used to investigate the protective effect of an organic thiophosphate, amifostine, against the toxicity of antracyclines. Premitotic DNA synthesis was found to be more sensitive than premeiotic DNA synthesis to antracyclines. Idarubicin was more toxic than doxorubicin to germ cells in inducing apoptosis and suppressing DNA synthesis. Amifostine had no protective effect against doxorubicin- or idarubicin-induced inhibition of DNA synthesis. In contrast, a significant stimulation of DNA synthesis in premitotic cells by amifostine was found, suggesting that this compound may have a stimulative effect on spermatogenic stem cells. These data show that stage-specific dissection of the seminiferous tubules and their in vitro exposure to predetermined doses of drugs may give us a unique possibility to detect drug action and protection against the cytotoxicity of antineoplastic agents at the cellular level of the spermatogenic cycle.

Parthenogenetic activation of porcine oocytes by using 7% ethanol, 50 or 100 μM A23187 results in an increase in intracellular pH as does prolonged exposure to thimerosal. We attempt to specify which transporters or mechanisms are involved in the observed increase in intracellular pH during oocyte activation. Experiments were performed in the absence of sodium; the presence of 2.5 mM amiloride, a potent inhibitor of the Na /H antiport; in the absence of bicarbonate; and in the presence of 4,4′-diisothiocyanatodihydrostilbene-2,2′-di-sulfonic acid, disodium salt (H₂DIDS) for all three activation methods. These treatments had no effect on the increase in intracellular pH induced by the calcium ionophore or thimerosal, but all reduced the increase in pH (P < 0.001) in the 7% ethanol group. This suggests that the Na /H antiport and the HCO₃⁻/Cl⁻ exchangers are not playing a role during treatment with calcium ionophore or thimerosal, and the pH increase observed during treatment with 7% ethanol may be dependent upon a sodium or bicarbonate flux (or both) into the oocyte. Bafilomycin A1 (500 nm), an inhibitor of vacuolar-type H ATPases, had no effect on 7% ethanol or thimerosal treatments, but significantly reduced the increase in intracellular pH observed during calcium ionophore treatment. This may be the result of an initial local increase in intracellular free calcium levels.

The main objective of the study was to investigate the effects of hyperthyroidism on the rat testis interstitium during prepuberty, which is not well understood at present. Male Sprague Dawley rats were injected subcutaneously daily with saline (controls) or tri-iodothyronine (T₃, 50 μg/kg body weight; hyperthyroids) from postnatal Day 1. Rats were killed at Days 5, 7, 9, 12, 16, and 21. One testis of each rat was used to determine LH-stimulated (100 ng/ml) testicular androgen secretory capacity in vitro. The other testis was used either for morphometric studies (n = 5) or for immunolocalization of 3β-hydroxysteroid dehydrogenase (3β-HSD) to identify steroidogenic cells (n = 3) and 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) to differentially identify adult Leydig cells. Daily T₃ injections resulted in significant reductions in body and testis weights. Morphometric analysis revealed that lower testis weights in rats treated with T₃ were mainly the result of reductions of total volume of seminiferous cords/tubules. The number of interstitial mesenchymal cells (MCs) was lower (P < 0.05) in T₃ rats compared with age-matched controls. The number of fetal Leydig cells (FLCs) was not different between the two groups; however, FLC hypotrophy was detected in T₃ rats at Day 16 in contrast to Day 21 in control rats. In both groups, morphologically identifiable adult Leydig cells (ALCs) were observed at Day 12 and thereafter; however, the ALC number per testis in T₃ rats was twice as much as those of controls. Positive immunolabeling for 3β-HSD was first detected in MC/progenitor cells on Day 9 in rats in the T₃ group (cells were still spindle-shaped) and on Day 12 in rats in the control group. Testicular testosterone production in vitro was lower (P < 0.05) in T₃ rats compared with controls at each age tested and further reductions (<0.05) were observed in T₃ rats at Days 16 and 21. Testicular androstenedione production was also lower (P < 0.05) in T₃ rats at Days 5 and 7, but increased (P < 0.05) thereafter, than in control rats. These findings support that there are more newly formed ALCs in T₃ testes than in those of controls. Moreover, these results demonstrate that hyperthyroidism stimulates premature hypotrophy of FLCs and early differentiation of increased numbers of MCs to ALCs in the prepubertal rat testis, further supporting the view that thyroid hormone has a regulatory role in initiating MC differentiation into ALCs in the prepubertal rat testis.

Interstitial cells in the neonatal hamster do not respond to LH in vitro; however, side-chain cleavage (CYP11A1) and 17α-hydroxylase (CYP17) enzyme proteins are expressed in these cells. The objective of the study was to evaluate whether the cAMP second messenger system was active in these cells and if cAMP upregulates the levels of CYP11A1 and CYP17 mRNA. Interstitial cells (ICs) were cultured for 96 h in the presence of 5% fetal bovine serum and then cultured in serum-free medium in the presence of LH, forskolin, or 8-Br-cAMP for 24 h. The accumulation of cAMP, progesterone, and androstenedione was measured by radioimmunoassay, whereas CYP11A1 and CYP17 mRNA levels were determined by a semiquantitative reverse transcription-polymerase chain reaction and Southern hybridization analysis. LH failed to induce either progesterone or androstenedione production; however, forskolin stimulated cAMP production by interstitial cells in a dose-dependent manner. Moreover, both forskolin and 8-Br-cAMP significantly elevated the levels of CYP11A1 and CYP17 mRNA and induced progesterone synthesis by the interstitial cell monolayer. Despite the increase in CYP17 mRNA levels by 8-Br-cAMP, no appreciable change was noted in androstenedione production. These results suggest that, in vitro, a fully functional adenylate cyclase system is present in cultured interstitial cells of the neonatal hamster and that cAMP can influence the expression of CYP11A1 and CYP17 genes; however, cultured cells do not appear to express LH receptors that are functionally linked to the adenylate cyclase system. Moreover, the translation of CYP17 mRNA may require additional factors, which may originate from maturing granulosa cells.

This goal of this study was to examine immunohistochemical distribution of leukemia inhibitory factor (LIF), LIF receptor (LIFR), and glycoprotein (gp) 130 in rhesus monkey uterus during the menstrual cycle and early pregnancy. Pregnancy rate was significantly reduced in the control group from 66.7% (12 of 18) to 22.2% (4 of 18) with an injection of goat anti-human recombinant LIF immunoglobulin G into the uterine lumen on Day 8 of pregnancy. LIF was mainly localized in glandular and luminal epithelium. LIF immunostaining during the luteal phase was stronger than it was during the proliferative phase. LIF staining gradually increased from Day 3 of pregnancy and reached its highest level on Day 9. LIFR was mainly localized in the glandular and luminal epithelium. LIFR staining during the luteal phase was stronger than it was during the proliferative phase. LIFR staining began to increase from Day 3 of pregnancy and reached a high level on Days 9 and 11. Gp130, a signal-transducing receptor component of LIF, was mainly localized in the glandular epithelium. A high level of gp130 was found on Days 16 and 20 of menstrual cycle, and from Days 5 to 11 of pregnancy. These results suggest that LIF may play an important role in monkey implantation, as it does in mice.

Successful cryopreservation of mammalian oocytes would provide a steady source of materials for nuclear transfer and in vitro embryo production. Our goal was to develop an effective vitrification protocol to cryopreserve bovine oocytes for research and practice of parthenogenetic activation, in vitro fertilization, and nuclear transfer. Bovine oocytes matured in vitro were placed in 4% ethylene glycol (EG) in TCM 199 plus 20% fetal bovine serum (FBS) at 39°C for 12–15 min, and then transferred to a vitrification solution (35% EG, 5% polyvinyl-pyrrolidone, 0.4 M trehalose in TCM 199 and 20% FBS). Oocytes were vitrified in microdrops on a precooled (−150°C) metal surface (solid-surface vitrification). The vitrified microdrops were stored in liquid nitrogen and were either immediately thawed or were thawed after storage for 2–3 wk. Surviving oocytes were subjected to 1) parthenogenetic activation, 2) in vitro fertilization, or 3) nuclear transfer with cultured adult fibroblast cells. Treated oocytes were cultured in KSOM containing BSA or FBS for 9 to 10 days. Embryo development rates were recorded daily and morphologically high-quality blastocysts were cryopreserved for nuclear transfer-derived embryos at Day 7 or Day 8 of culture. Immediate survival of vitrified/thawed oocytes varied between 77% and 86%. Cleavage and blastocyst development rates of vitrified oocytes following in vitro fertilization or activation were lower than those of the controls. For nuclear transfer, however, vitrified oocytes supported embryonic development as equally well as fresh oocytes.

An expressed-sequence tag database search has identified three rat cDNA clones in the prolactin/growth hormone family, including a homologue of mouse proliferin-related protein (PRP). The encoded proteins of the two novel clones, designated prolactin-like proteins L (PLP-L) and M (PLP-M), are predicted to be synthesized as precursors of 229 and 227 amino acids, modified by N-linked glycosylation, and secreted as mature glycoproteins of 199 and 200 residues, respectively. Murine homologues to PLP-L and PLP-M were also identified. The open reading frame of rat PRP encodes a precursor protein of 245 amino acids and predicts a secreted 215-amino acid glycoprotein with 81% identity to mouse PRP. All three rat mRNAs are expressed in the placenta, and expression is not detected in other tissues. PLP-L mRNA expression is observed from Days 11–20, with highest levels at Day 13; highest levels of PLP-M are observed from Day 11 until parturition, with peak levels also on Day 13; and highest levels of PRP are also observed from Day 11 until term, with maximal expression on Day 17. All three genes are most highly expressed in invasive trophoblast cells lining the central placental vessel. The identification of molecular markers for endovascular trophoblasts serves to highlight the invasive nature of rodent placentation and may prove useful for future studies of placental function.

Little is known about the reproductive biology of Australia's critically endangered northern hairy-nosed wombat (Lasiorhinus krefftii), largely due to its cryptic nature and the difficulty in accessing the small remaining population of about 70 animals. Using the noninvasive technique of fecal steroid analysis, we have examined the endocrinology of the more common yet closely related southern hairy-nosed wombat (Lasiorhinus latifrons). The aims of this study were to 1) develop and validate fecal androgen analysis in this species, 2) examine and compare seasonal differences in fecal and plasma androgens in male wombats, and 3) correlate seasonal differences in androgens with changes in male accessory glands (prostate and bulbourethral gland). Fecal androgens were extracted in ether; concentrated; separated by HPLC into testosterone (T), dihydrotestosterone (DHT), and 5α-androstane-3α,17β-diol (Adiol) fractions; and quantitated by RIA. The concentrations of androgens in fecal pellets from 14 wild southern hairy-nosed wombats as determined by RIA varied over the range 6.6–25.0 ng/g dry weight for T, 4.0–24.2 ng/g dry weight for DHT, and 0–34.8 ng/g dry weight for Adiol. For each androgen, a highly significant linear correlation was observed between plasma and fecal concentrations. When individuals were grouped into either breeding season (pellets collected between August–November) or nonbreeding season (collected between February–April), significant (P < 0.05) differences between seasons were observed for both plasma and fecal T, plasma DHT, and fecal Adiol. For all androgens, the mean fecal and plasma concentrations were higher during the breeding season than the nonbreeding season. A significant (P < 0.001) correlation was observed between fecal T and prostate weight, while DHT and Adiol correlations were nonsignificant. Significant correlations were observed, however, between all three fecal androgens and bulbourethral gland weight. These studies demonstrate that fecal T is a valid indicator of reproductive status in the male southern hairy-nosed wombat, with significant correlations observed between fecal T, plasma T, and prostate and bulbourethral gland weights. These findings have important implications for the study of the reproductive endocrinology of the critically endangered northern hairy-nosed wombat.

The regulation of the phospholipase C (PLC) and the expression of inositol 1,4,5-trisphosphate receptors (IP₃Rs) in terms of mRNA, proteins, and binding capacity were examined in the rat myometrium and endometrium at midgestation (Day 12) and at term (Day 21) comparatively to the estrogen-treated tissues (Day 0). In both uterine tissues, the production of inositol phosphates mediated by carbachol as well as by AlF₄⁻ was enhanced with advancing gestation. ³[H]IP₃ binding sites in membranes also increased during pregnancy (Day 21 > Day 12 > Day 0). The mRNAs encoding for three isoforms of IP₃R as well as their corresponding proteins, IP₃R-1, IP₃R-2, and IP₃R-3 were coexpressed, albeit to different extents, in the myometrium and endometrium. The expression of IP₃Rs increased with advancing gestation, except for IP₃R-2 that increased only in the endometrium at term. Thus, the pregnancy-related upregulation of the PLC cascade coincided with an increase in the expression of IP₃Rs. The difference noted between the two uterine tissues suggests that IP₃Rs may have cell-specific functions.

The present study tested the hypothesis that acute treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) impairs fertility, disrupts the nocturnal melatonin rhythm, and suppresses lymphocyte function. Adult Siberian hamsters administered 2 or 100 μg TCDD/kg body weight/0.2 ml sesame oil had a delayed latency to first litter and an increased adult mortality compared to hamsters given 0.1 μg/kg or vehicle. Within 75 days of TCDD treatment, full reproductive capabilities were achieved. Moreover, the nocturnal melatonin rhythm was not disrupted in adults administered TCDD or in their progeny. Lymphocyte activity varied with respect to time of day and treatment. Lymphocyte proliferation was enhanced at night irrespective of TCDD treatment; during the day, 2 wk after the 2-μg/kg treatment, blastogenesis was reduced compared to that in the 0.1-μg/kg group or in vehicle-treated controls. In contrast, TCDD did not affect the mixed lymphocyte reaction in response to allogeneic antigen when assessed at 2 and 20 wk post-treatment. Thus, findings indicate that TCDD produced acute effects on fertility, mortality, and systemic lymphocyte proliferation, but long-lasting effects on specific aspects of reproductive, neuroendocrine, and immune cell functions were not observed.

Tissue type (t) and urokinase type (u) plasminogen activators (PAs) have been shown to be secreted by Sertoli cells in the seminiferous tubules in a cyclic fashion and to be dependent upon FSH stimulation or upon the presence of adjacent spermatogenic cells. In the present study we have analyzed the production of PAs by retinoid-treated rat Sertoli cells. In addition, because retinoids modulate the response of Sertoli cells to FSH either potentiating or antagonizing its action, we have investigated a possible modulation of FSH-stimulated PA production. Under basal conditions, Sertoli cells, isolated from prepubertal rats, secrete predominantly uPA. A significant dose-dependent inhibition of uPA activity was observed after treatment with retinol, while no significant effect was detected upon tPA secretion. When Sertoli cells were cultured in the presence of 0.25 μM retinol, a significant inhibition of uPA activity was evident after 16 h of treatment and reached approximately 80% after 48 h of treatment. The analysis of the mRNA levels revealed that retinol induces an inhibition of the steady-state levels of uPA mRNA without affecting those of tPA. Moreover, retinol affected uPA mRNA levels by increasing mRNA turnover. The effect of retinoids on Sertoli cells isolated from older animals was less evident, possibly due to the reduced production of uPA with the increase of age of the donor animals. Our results on the effect of retinoids upon Sertoli cell uPA production reinforce the importance of retinoids in the control of postnatal testis development.

Effects of microgravity (μG) on fertilization were studied in the urodele amphibian Pleurodeles waltl on board the MIR space station. Genetic and cytomorphologic analyses ruled out parthenogenesis or gynogenesis and proved that fertilization did occur in μG. Actual fertilization was demonstrated by the analysis of the distribution of peptidase-1 genes, a polymorphic sex-linked enzyme, in progenies obtained in μG. Further evidence of fertilization was provided by the presence of spermatozoa in the perivitelline space and in the fertilization layer of the μG eggs and by the presence of a female pronucleus and male pronuclei in the egg cytoplasm. Experiments in μG and in 1.4G, 2G, and 3G hypergravity showed for the first time that, compared to eggs in 1G, several characteristics of the fertilization process including the cortical reaction and the microvillus transformations were altered depending on the gravitational force applied to the eggs. Microvillus elevation, the most evident feature, was reduced on μG-eggs and amplified on eggs submitted to 2G and 3G. No lethal consequences of these alterations on the early development of μG-eggs were observed.

Placental hypoxia likely plays an important role in both normal placental development and pathology. Yet, the molecular mechanisms of hypoxia signaling in this organ are virtually unexplored. Therefore, we investigated the expression of the hypoxia inducible transcription factors (HIF) in normal human placentas spanning the first trimester to term. Several key observations emerged: 1) HIF-1α and -2α mRNA were present in placentas of all gestational ages but with greater variability during early pregnancy; 2) overall, HIF-1α mRNA was expressed at a constant level in all placentas, whereas HIF-2α mRNA increased significantly with gestational age; 3) both HIF-1α and -2α protein decreased significantly with gestational age; and 4) HIF-1α and -2α immunoreactivity were overlapping in cellular distribution being expressed by the syncytiotrophoblast, villous cytotrophoblast, and fetoplacental vasculature with both nuclear and cytoplasmic localization. Next, we studied the regulation of these transcription factors by oxygen using placental villous explants in culture from first-trimester and term placentas. The major findings were 1) HIF-1α and -2α protein, but not mRNA, was induced by hypoxia in the placental villous explants; 2) HIF-1α DNA-binding activity was also stimulated by hypoxia; and 3) glucose transporter-1 mRNA (a known target of HIF) was also increased by hypoxia in placental villous explants. We suggest that physiological hypoxia contributes to the increased expression of HIF-1α and -2α protein in early placentas and that regulation of these transcription factors by hypoxia in the human placenta occurs at the level of protein and not mRNA.

An analysis of the pattern of expression of the mouse placental hormone prolactin-like protein A (PLP-A) has revealed that this hormone is expressed exclusively in secondary trophoblast giant cells but not in primary giant cells. Thus, PLP-A serves as a marker for a subset of giant cells. Recent results have indicated that PLP-A binds to and inhibits the activity of natural killer cells, and thus, the localized expression of PLP-A may be important for regulating the activity of this class of T lymphocytes in a restricted region of the implantation site. Previous studies indicated that the transcription factor GATA-2 is required for the trophoblast giant cell-specific expression of two other hormones in the prolactin family, placental lactogen I and proliferin. In the absence of GATA-2, PLP-A continues to be expressed, but in this mutant background, PLP-A mRNA is detected in both primary and secondary giant cells. Thus, GATA-2 contributes both to positive and negative regulation of trophoblast giant cell-specific gene expression, and this factor apparently plays an important role in generating or maintaining the distinct functions of secondary, compared with primary, trophoblast giant cells.

The fertilization-induced exocytosis of egg cortical granules (CGs) is responsible for a block to polyspermy, crucial to the viability of many species. The contents of mammalian CGs have been an elusive target for analysis because of picogram quantities of CG proteins. By using media enriched in secreted CG contents from calcium ionophore-induced eggs as an immunogen, a monoclonal antibody was raised that immunolocalized to structures in the mouse egg cortex with all the hallmarks of CGs. These structures were the correct size, absent from the region over the metaphase II spindle, and greatly reduced after fertilization. Double-labeling experiments confirmed that the antibody recognized the same population of CGs as those recognized by Lens culinaris agglutinin. On Western blots, the antibody primarily recognized a 32-kDa protein (and secondarily one at ∼25 kDa) in mouse eggs. Analysis of biotin-labeled secreted proteins from activated eggs confirmed that CGs release only a small number of major proteins (45, 34, 32, 28, and ∼20 kDa by SDS-PAGE). We therefore propose that the 32-kDa protein identified by this antibody is likely to correspond to the 32-kDa protein released from activated eggs and that it may be involved in the block to polyspermy. These methods should make it possible to generate additional antibodies to study the structure of CG components as well as their roles in the polyspermy block and CG biogenesis.

The strictly maternal inheritance of mitochondria and mitochondrial DNA (mtDNA) in mammals is a developmental paradox promoted by an unknown mechanism responsible for the destruction of the sperm mitochondria shortly after fertilization. We have recently reported that the sperm mitochondria are ubiquitinated inside the oocyte cytoplasm and later subjected to proteolysis during preimplantation development (P. Sutovsky et al., Nature 1999; 402:371–372). Here, we provide further evidence for this process by showing that the proteolytic destruction of bull sperm mitochondria inside cow egg cytoplasm depends upon the activity of the universal proteolytic marker, ubiquitin, and the lysosomal apparatus of the egg. Binding of ubiquitin to sperm mitochondria was visualized by monospecific antibodies throughout pronuclear development and during the first embryonic divisions. The recognition and disposal of the ubiquitinated sperm mitochondria was prevented by the microinjection of anti-ubiquitin antibodies and by the treatment of the fertilized zygotes with lysosomotropic agent ammonium chloride. The postfecundal ubiquitination of sperm mitochondria and their destruction was not seen in the hybrid embryos created using cow eggs and sperm of wild cattle, gaur, thus supporting the hypothesis that sperm mitochondrion destruction is species specific. The initial ligation of ubiquitin molecules to sperm mitochondrial membrane proteins, one of which could be prohibitin, occurs during spermatogenesis. Even though the ubiquitin cross-reactivity was transiently lost from the sperm mitochondria during epididymal passage, likely as a result of disulfide bond cross-linking, it was restored and amplified after fertilization. Ubiquitination therefore may represent a mechanism for the elimination of paternal mitochondria during fertilization. Our data have important implications for anthropology, treatment of mitochondrial disorders, and for the new methods of assisted procreation, such as cloning, oocyte cytoplasm donation, and intracytoplasmic sperm injection.

Telomeres, the noncoding sequences at the ends of chromosomes, progressively shorten with each cellular division. Spermatozoa have very long telomeres but they lack telomerase enzymatic activity that is necessary for de novo synthesis and addition of telomeres. We performed a telomere restriction fragment analysis to compare the telomere lengths in immature rat testis (containing type A spermatogonia) with adult rat testis (containing more differentiated germ cells). Mean telomere length in the immature testis was significantly shorter in comparison to adult testis, suggesting that type A spermatogonia probably have shorter telomeres than more differentiated germ cells. Then, we isolated type A spermatogonia from immature testis, and pachytene spermatocytes and round spermatids from adult testis. Pachytene spermatocytes exhibited longer telomeres compared to type A spermatogonia. Surprisingly, although statistically not significant, round spermatids showed a decrease in telomere length. Epididymal spermatozoa exhibited the longest mean telomere length. In marked contrast, telomerase activity, measured by the telomeric repeat amplification protocol was very high in type A spermatogonia, decreased in pachytene spermatocytes and round spermatids, and was totally absent in epididymal spermatozoa. In summary, these results indicate that telomere length increases during the development of male germ cells from spermatogonia to spermatozoa and is inversely correlated with the expression of telomerase activity.

In rats, an acidic luminal pH maintains sperm quiescence during storage in the epididymis. We recently showed that vacuolar H ATPase-rich cells in the epididymis and vas deferens are involved in the acidification of these segments. Treatment of rats with cadmium (Cd) leads to alkalinization of this fluid by an unknown mechanism. Because Cd may affect H ATPase function, we examined 1) the in vivo effect of Cd poisoning on H ATPase-rich cell morphology and on the abundance and distribution of the 31-kDa H ATPase subunit in cells along the rat epididymis, and 2) the in vitro effect of Cd on H ATPase activity and function in the isolated vas deferens. Immunofluorescence and immunoblotting data from rats treated with Cd for 14–15 days (2 mg Cd/kg body mass/day) showed that 1) H ATPase-positive cells regressed to a prepubertal phenotype, and 2) H ATPase was lost from the apical pole of the cell and was redistributed into an intracellular compartment. In experiments in vitro, Cd inhibited bafilomycin-sensitive ATPase activity in isolated total cell membranes and, as measured using a proton-selective extracellular microelectrode, inhibited proton secretion in isolated vas deferens. We conclude that alkalinization of the tubule fluid in the epididymis and vas deferens of Cd-treated rats may result from the loss of functional H ATPase enzyme in the cell apical domain as well as from a direct inhibition of H ATPase function by Cd.

Using rapid amplification of cDNA ends, a cDNA encoding a novel splice variant of the human Cα catalytic subunit of cAMP-dependent protein kinase (PKA) was identified. The novel isoform differed only in the N-terminal part of the deduced amino acid sequence, corresponding to the part encoded by exon 1 in the previously characterized murine Cα gene. Sequence comparison revealed similarity to an ovine Cα variant characterized by protein purification and micropeptide sequencing, Cα-s, identifying the cloned human cDNA as the Cα-s isoform. The Cα-s mRNA was expressed exclusively in human testis and expression in isolated human pachytene spermatocytes was demonstrated. The Cα-s protein was present in ejaculated human sperm, and immunofluorescent labeling with a Cα-s-specific antibody indicated that Cα-s was localized in the midpiece region of the spermatozoon. The majority of Cα-s was particulate and could not be released from the sperm midpiece by cAMP treatment alone. Furthermore, detergent extraction solubilized approximately two-thirds of the Cα-s pool, indicating interaction both with detergent-resistant cytoskeletal and membrane structures. In addition, we recently identified the regulatory subunit isoforms RIα, RIIα, and an A-kinase anchoring protein, hAKAP220 in this region in sperm that could target Cα-s. This novel Cα-s splice variant appeared to have an independent anchor in the human sperm midpiece as it could not be completely solubilized even in the presence of both detergent and cAMP.

Male homozygous transgenic c-ros knockout mice are sterile by natural mating, lack a part of their epididymis, and the epididymal sperm exhibit tail angulation in vivo and in vitro. To ascertain if this abnormal tail form caused the infertility, the number and nature of sperm in the tract of females mated to knockout and wild-type mice were determined. Percentage motility and numbers of sperm in the uterus 1 h after mating were similar between genotypes. The majority of the uterine sperm from the wild-type males had straight flagella, whereas 46–86% of knockout sperm were bent at the cytoplasmic droplet even when motile. Motile knockout sperm showed a 54 and 37% reduction in the straightline and curvilinear velocities compared with straight wild-type sperm. Sequential flushings of the oviduct 4 h after mating with the wild-type males contained sperm: 591 ± 119 free, 371 ± 70 loosely, and 122 ± 47 tightly bound to the epithelium, but no knockout sperm were recovered from the oviduct or observed within the uterotubal junction in tissue sections. The infertility of c-ros knockout male mice can be explained by the sperm's inability to enter the oviduct, as a result of their bent tails forming the entangled sperm mass and their compromised flagellar vigor within the uterus.

An interferon (IFN)-stimulated gene (ISG) encodes a bovine 17-kDa protein (bISG17) that is released from endometrial cells but also conjugates to intracellular proteins through a ubiquitinlike mechanism. During early pregnancy in ruminants, conceptus-derived IFN-τ induces endometrial ISG17. The present experiments were designed to generate bioactive recombinant (r) bISG17. The Pichia pastoris yeast expression system was used because previous experiments expressing the human ISG15 ortholog in bacteria were confounded by inherent carboxypeptidase activity that cleaved C-terminal residues resulting in an inactive protein. In a series of extensive yeast culture experiments using shaker-bath and fermentation approaches, optimal conditions were determined for a transformant containing a multi-ISG17 gene insertion. Recombinant bISG17 was purified. Carboxy-terminal sequencing revealed that rbISG17 retained the C-terminal Gly that is potentially critical for the first step in covalent attachment to targeted intracellular proteins. The rISG17 induced (P < 0.0001) IFN-γ mRNA (reverse transcription–polymerase chain reaction) and release of IFN-γ protein (ELISA) by bovine peripheral blood mononuclear cells. The IFN-γ mRNA also was upregulated (P < 0.0001) in endometrium from pregnant (Day 18) when compared with nonpregnant (Days 14 and 18) cows. It is concluded that rbISG17 generated in a yeast expression system retains cytokine/hormonal activity. This is the first description coupling the biology of two distinct IFNs (γ and τ) through the intermediary ubiquitin homolog ISG17.

The sperm head of the plains rat, an Australian hydromyine rodent, is highly complex in structure and contains, in addition to an apical hook, two large ventral processes (VPs) that extend from its upper concave surface and that are largely composed of a huge extension of the sperm head cytoskeleton surrounded by postacrosomal dense lamina. In this study we have attempted to determine their protein composition. For this, the VPs were isolated, the proteins within them separated by SDS-PAGE, and the resultant polypeptide bands Western blotted and probed with antibodies against laboratory rat perforatorial and bull perinuclear theca sperm proteins. Antibodies were also used to determine the perforatorial and perinuclear theca proteins by immunogold labeling of transmission electron microscopic sections. The results indicate that the material within the VPs is largely composed of perforatorial cross-reacting proteins together with F-actin with the dominant protein being PERF 15. The perinuclear theca proteins are, by contrast, restricted to a narrow region adjacent to the acrosomal and nuclear membranes. In conclusion, this study has shown that the VPs of the spermatozoa of Australian rodents are perforatorial-like appendages that contain similar proteins to the perforatorium of the apical hook together with F-actin; their functional significance remains unknown.

The mechanism by which GnRH increases sperm-zona pellucida binding in humans was investigated in this study. We tested whether GnRH increases sperm-zona binding in Ca² -free medium and in the presence of Ca² channel antagonists. We also examined the GnRH effect on the intracellular free Ca² concentration ([Ca² ]_i). Sperm treatment with GnRH increased sperm-zona binding 300% but only when Ca² was present in the medium. In Ca² -free medium or in the presence of 400 nM nifedipine, 80 μM diltiazem, or 50 μM verapamil, GnRH did not influence sperm-zona binding. GnRH increased the [Ca² ]_i in the sperm in a dose-dependent manner. The maximum effect was reached with 75 nM GnRH. The GnRH-induced increase in [Ca² ]_i was fast and transient, from a basal [Ca² ]_i of 413 ± 22 nM to a peak value of 797 ± 24 nM. The GnRH-induced increase in [Ca² ]_i was entirely due to a Ca² influx from the extracellular medium because the increase in [Ca² ]_i was blocked by the Ca² chelator EGTA and by the Ca² channel antagonists nifedipine and diltiazem. These antagonists, however, were not able to inhibit the progesterone-activated Ca² influx. On the contrary, T-type calcium channel antagonists pimozide and mibefradil did not affect GnRH-activated Ca² influx but inhibited the progesterone-activated Ca² influx. Finally, the GnRH-induced Ca² influx was blocked by two specific GnRH antagonists, Ac-D-Nal¹-Cl-D-Phe²-3-Pyr-D-Ala³-Arg⁵-D-Glu(AA)⁶-GnRH and Ac-^3,4-dehydro-Pro¹,-p-fluoro-D-Phe², D-Trp^3,6-GnRH. These results suggest that GnRH increases sperm-zona binding via an elevation of [Ca² ]_i through T-type, voltage-operated calcium channels.

The synaptosome-associated protein of 25 kDa (SNAP-25) is crucially involved in exocytosis in neurons. The aim of this study was to investigate whether it is present in the ovary. We found SNAP-25 to be expressed in nonneuronal cells of the rat and human ovary, namely in all oocytes and in steroidogenic cells, including granulosa cells (GC) of large antral follicles and luteal cells. Both isoforms, SNAP-25a and b, were found in the ovary. Oocytes obtained by laser capture microdissection were shown to express SNAP-25b, whereas SNAP-25a was found in rat GC and human luteinized GC. Immunohistochemical observations of strong SNAP-25 staining in GC of large growing antral follicles compared with absent or weak staining in small follicles suggested a role in folliculogenesis. To study a presumed regulation of SNAP-25, we used a rat GC line (GFSHR-17), which expresses FSH receptors, and luteinizing human GC, which express LH receptors. FSH elevated SNAP-25 mRNA and protein levels about fivefold within 24 h in GFSHR-17 cells. The cAMP analogue dibutyryl-cAMP (db-cAMP) mimicked this action of FSH. The effects of both db-cAMP and FSH were inhibited by the protein kinase A (PKA) inhibitor H89. In contrast, SNAP-25 protein and mRNA-levels were not altered by LH/hCG in luteinized human GC. Our results for the first time identify SNAP-25b in oocytes and SNAP-25a in steroidogenic cells of the mammalian ovary. SNAP-25a and b may be involved in different exocytotic processes in these cell types.

Previous studies have demonstrated that cGMP is produced by nitric oxide-mediated activation of soluble guanylyl cyclase (sGC) in seminiferous tubules of the human testis. It is not known, however, whether carbon monoxide (CO), another activator of sGC, is also involved in testicular function. To address this issue, testicular probes from 65- to 75-yr-old men have been examined. The CO-generating enzyme, heme oxygenase-1 (HO-1), could be localized by immunohistochemical and immunoblot analyses to Sertoli cells. In these cells, HO-1 is detectable in adluminal cell compartments, whereas sGC immunoreactivity is distributed exclusively in basal compartments. Treatments of isolated tubules with either sodium arsenite, known to induce HO-1, or hematin, an HO substrate, resulted in 4.4- and 1.8-fold, respectively, increases in cGMP levels. ODQ, a specific sGC inhibitor, inhibited completely the sodium arsenite-stimulated cGMP production. Moreover, the HO inhibitor zinc protoporphyrin-IX and the CO scavenger hemoglobin both significantly reduced (77% or 46% of control, respectively) tubular cGMP generation. These findings, demonstrating for the first time a link between HO-1 activity in Sertoli cells and sGC-dependent cGMP production in seminiferous tubules, suggest a functional role of CO in the human testis.

Fully grown competent mouse oocytes spontaneously resume meiosis in vitro when released from their follicular environment, in contrast to growing incompetent oocytes, which remain blocked in prophase I. The cell cycle regulators, maturation promoting factor (MPF; [p34^cdc2/cyclin B kinase]) and mitogen-activated protein (MAP) kinases (p42^MAPK and p44^MAPK), are implicated in meiotic competence acquisition. Incompetent oocytes contain levels of p42^MAPK, p44^MAPK, and cyclin B proteins that are comparable to those in competent oocytes, but their level of p34^cdc2 is markedly lower. Okadaic acid (OA), an inhibitor of phosphatases 1 and 2A, induces meiotic resumption of incompetent oocytes. The kinetics and the percentage of germinal vesicle breakdown depends on whether or not oocytes have been cultured before OA treatment. We show that the fast kinetics and the high percentage of germinal vesicle breakdown induced by OA following 2 days in culture is neither the result of an accumulation of p34^cdc2 protein, nor to the activation of MPF in incompetent oocytes, but rather by the premature activation of MAP kinases. Indeed, a specific inhibitor of MAPK kinase (MEK) activity, PD98059, inhibits activation of MAP kinases and meiotic resumption. Altogether, these results indicate that the MEK-MAPK pathway is implicated in OA-induced meiotic resumption of incompetent mouse oocytes, and that the MEK-MAPK pathway can induce meiotic resumption in the absence of MPF activation.