At the 1999 annual meeting of the Society for the Study of Reproduction there were three speakers in the minisymposium entitled ``I've got to get out of here: fetal-maternal interactions involved in parturition''. The primary focus was on research progress in understanding the mechanisms involved in human parturition. Although the title of the symposium emphasized the need to ``get out'', there was considerable emphasis on understanding the problem of ``getting out too early'' or preterm birth. While preterm birth is unusual in most species, it is of major clinical importance in the human. The data presented by one of the speakers is reviewed here with a focus on preterm labor and preterm premature rupture of the fetal membranes as mechanisms involved in the diverse pathology of preterm birth.

Follicle development is the result of a balanced ratio between cell proliferation and cell death. Previous studies demonstrated differential mitotic responses to insulin-like growth factor (IGF)-I and epidermal growth factor (EGF) of cumulus cells (CC) and mural granulosa cells (MGC). Because cell-to-cell contact seems to modulate the occurrence of programmed cell death, the present experiments investigated the role of cell association in mediating apoptosis and the mitogenic responses to these growth factors of CC and MGC. Cumulus cells were cultured either as intact cumulus-oocyte complexes (COC) or after dissociation with EGTA sucrose, in the presence of 50 ng/ml IGF-I, 5 ng/ml EGF, or both. Mural granulosa cells from the same follicles were similarly cultured either as cell aggregates or as dissociated cells. Synthesis of DNA was assessed by measurement of [³H]thymidine incorporation during the last 6 h of a 24-h culture in TCM199. Percentages of cells undergoing apoptosis were determined immunohistochemically in intact COC and GC aggregates, before and after dissociation as well as after the culture period. Epidermal growth factor and IGF-I stimulated DNA synthesis in both cell types; however, EGF inhibited the action of IGF-I in intact COC but not in MGC. Compared to nondissociated cells, dissociation resulted in a reduction of the mitogenic response of CC to both growth factors and of MGC to EGF. Unlike the response of intact COC to combined treatment with the two growth factors, dissociated CC displayed additive responses to the two growth factors in combination. Addition of denuded oocytes to cultures of dissociated CC enhanced both basal and growth factor-stimulated DNA synthesis but did not restore the inhibitory effect of EGF on the IGF-I response characteristic of intact COC. A significant proportion of intact MGC aggregates underwent apoptosis after 24 h of culture, while no increase of apoptotic cells was observed in intact COC. A dramatic increase in the percentage of apoptotic cells was observed in both CC and MGC when cell-cell contact was interrupted, and EGF and IGF-I were able to partially prevent its occurrence. Taken together these data showed that CC and MGC exhibit qualitatively and quantitatively different responses to IGF-I when cultured in the presence of EGF both in terms of DNA synthesis and onset of apoptosis. Moreover, the disruption of cell-cell contact was a major factor reducing cell proliferation and inducing apoptosis among both subsets of GC.

The temporal and spatial expression of cytochrome P450 side-chain cleavage (CYP11A1) and cytochrome P450 17α-hydroxylase (CYP17) mRNA and protein during thecal cell differentiation in developing hamster ovaries were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence histochemistry, respectively. Ovaries were collected from 15-day fetal through 20-day-old postnatal hamsters and used either for immunofluorescence detection of enzyme protein or RT-PCR evaluation of enzyme mRNA. Immunoreactivity of CYP11A1 first appeared in the interstitial cells on Day 10 postnatal (PN), and the intensity increased significantly with further ovarian development beyond 11 days of age. In contrast, CYP17 immunostaining was first detected in a few interstitial cells closer to large preantral follicles by Day 12 PN, and their number increased appreciably by Day 14 PN. By age 18–20 days, CYP17-positive cells were localized primarily in the thecal layer of large preantral follicles. A low level of CYP17 and CYP11A1 mRNA was present in fetal ovaries. The CYP17 mRNA levels increased sharply by Day 1 PN but decreased to a low baseline level by Day 2 PN and remained low up to Day 9 PN. Both CYP11A1 and CYP17 mRNA levels increased significantly by Day 10 PN compared to Day 9 PN; however, the increase for CYP11A1 was greater than CYP17. The CYP11A1 mRNA levels decreased noticeably on Day 11 PN and remained relatively stable until Day 14 PN; however, mRNA levels started increasing by Day 15 PN and increased sharply by Day 17 PN onward, corresponding to the increase in CYP11A1 protein in the ovarian interstitium and thecal compartments. On the other hand, CYP17 mRNA expression increased progressively through Day 12 PN. A sharp increase in CYP17 mRNA was noted on Day 13 PN in conjunction with the morphological development of thecal cells; mRNA levels remained steady afterward. The correlation of the increase in enzyme mRNA and protein, especially of CYP17, with the morphological development of thecal layers suggests that the differentiation of interstitial cells into theca may be modulated by multilayered preantral follicles, and the expression of enzyme protein occurs prior to an increase in serum LH.

Multiple isoforms of calpastatin have been identified with unique N-terminal regions followed by identical calpain inhibitory domains (II–IV). In many instances the isoforms are cell-type specific, although the precise functional differences among these N-terminal regions are largely unknown. Here we report a germ cell-specific isoform of calpastatin (tCAST) that consists of a novel N-terminal peptide of 40 amino acids (domain T) followed by domains II to IV of somatic calpastatin (sCAST). Domain T is responsible for membrane association of tCAST through a protein modification by myristylation. Mutation of the myristylation site eliminates membrane targeting. Unlike most of the isoforms of calpastatin that are generated through alternative RNA splicing or post-translational proteolysis, the testis-specific isoform is transcribed from an intronic promoter in haploid germ cells of the testis. The intronic promoter directs specific expression of a reporter transgene in developing germ cells of the mouse testis.

We have isolated a cDNA clone encoding a mouse haploid germ cell-specific protein from a subtracted cDNA library. Sequence analysis of the cDNA revealed high homology with pig and human heart succinyl CoA:3-oxo acid CoA transferase (EC 2.8.3.5), which is a key enzyme for energy metabolism of ketone bodies. The deduced protein consists of 520 amino acid residues, including glutamate 344, known to be the catalytic residue in the active site of pig heart CoA transferase and the expected mitochondrial targeting sequence enriched with Arg, Leu, and Ser in the N-terminal region. Thus, we termed this gene scot-t (testis-specific succinyl CoA:3-oxo acid CoA transferase). Northern blot analysis, in situ hybridization, and Western blot analysis demonstrated a unique expression pattern of the mRNA with rapid translation exclusively in late spermatids. The scot-t protein was detected first in elongated spermatids at step 8 or 9 as faint signals and gradually accumulated during spermiogenesis. It was also detected in the midpiece of spermatozoa by immunohistochemistry. The results suggest that the scot-t protein plays important roles in the energy metabolism of spermatozoa.

M-Phase promoting factor (MPF) is a complex of p34^cdc2 and cyclin B. Results of previous studies in which relative mass amounts of these cell cycle regulators were determined suggested that the accumulation of p34^cdc2, rather than cyclin B, could be a limiting factor in the acquisition of meiotic competence in mouse oocytes. Nevertheless, in the absence of measurements of the absolute amount of these components of MPF, it is possible that the molar amount of p34^cdc2 is in excess to that of cyclin B, i.e., the accumulation of p34^cdc2 is not a limiting factor. We report measurements of the absolute mass of p34^cdc2 and cyclin B1, as well as the two proximal regulators of MPF, namely cdc25C and wee1, in meiotically incompetent and competent mouse oocytes. We find that the numbers of molecules of p34^cdc2, cyclin B1, cdc25C, and wee1 in meiotically incompetent oocytes are 1.4 × 10⁶, 11.3 × 10⁶, 24.6 × 10⁶, 15.6 × 10⁶, respectively, and in meiotically competent oocytes the numbers are 14.3 × 10⁶, 95.5 × 10⁶, 80.0 × 10⁶, 40.1 × 10⁶, respectively. Thus, the concentration of cyclin B1 is always in excess to that of p34^cdc2, and this is consistent with the hypothesis that the accumulation of p34^cdc2 plays a role in the acquisition of meiotic competence. Last, the concentration of cdc25C is greater than that of wee1 and the concentration of each is greater than that of p34^cdc2 in both meiotically incompetent and competent oocytes.

The current study examines the expression and potential actions of neurotropin-3 (NT3), nerve growth factor (NGF), and their receptors during morphological sex determination (seminiferous cord formation) and perinatal rat testis development. The expression of neurotropins and their receptors was analyzed with immunohistochemistry. Cellular localization of neurotropin ligand and receptor proteins changed during embryonic testis development. Neurotropin-3 was localized to Sertoli cells at Embryonic Day 14 (E14), was present in gonocytes at Postnatal Day 0 (P0), and after birth became localized to the interstitium and Sertoli cells (P3–P5). The expression of trk C (the high affinity receptor for NT3) was localized to mesonephric ducts and cells surrounding the cords (E14–E18). In addition, Sertoli cells and preperitubular cells surrounding the cords at E14 also stained for trk C. Neurotropin-3 was expressed in gonocytes and Sertoli cells at P0–P5. Nerve growth factor was detected in Sertoli cells at E14, was clearly in Sertoli and interstitial cells at E16 and E18, and in Sertoli, germ, and interstitial cells from P0–P5. The expression of trk A (the high affinity receptor for NGF) was located in Sertoli and interstitial cells at E16–P5. To determine the actions of neurotropins during embryonic and perinatal testis development, experiments were conducted on E13 and P0 testis. Antisense oligonucleotide experiments with NT3 were used on E13 testis organ cultures to determine effects on seminiferous cord formation. Cord formation was inhibited in 40% of the organ cultures treated with the antisense NT3 oligonucleotides, while no inhibition was observed with sense oligonucleotides. In P0 testis cultures, both NT3 and NGF alone and in combination stimulated thymidine incorporation into DNA. Therefore, the neurotropins are involved in embryonic morphological events (cord formation; NT3) and in growth of the perinatal testis (P0; NT3 and NGF). To define further the growth effects of neurotropins on testis development, expression of transforming growth factor alpha and beta (TGFα and TGFβ) were examined in response to neurotropins. The P0 testis cultures were treated with neurotropins, and expression of mRNA for TGFα and TGFβ was analyzed utilizing a quantitative reverse transcription-polymerase chain reaction assay. Nerve growth factor and NT3 alone or in combination inhibited expression of mRNA for TGFα while NT3 increased mRNA expression of epidermal growth factor receptor. The combination treatment of neurotropins inhibited expression of TGFβ1 and increased expression of TGFβ3. In summary, observations suggest that NT3, NGF, trk A, and trk C are localized to cells critical to seminiferous cord formation and appear to be important regulators of morphological sex determination. In addition to these morphological effects, both NT3 and NGF stimulate P0 testis growth and may elicit their action through altering the expression of locally produced growth factors such as TGFα and TGFβ. Taken together these results suggest that neurotropins are regulators of paracrine cell-cell interactions that result in morphological sex determination and perinatal testis growth.

Histometrical evaluation of the testis was performed in 36 Piau pigs from birth to 16 mo of age to investigate Sertoli cell, Leydig cell, and germ cell proliferation. In addition, blood samples were taken in seven animals from 1 wk of age to adulthood to measure plasma levels of FSH and testosterone. Sertoli cell proliferation in pigs shows two distinct phases. The first occurs between birth and 1 mo of age, when the number of Sertoli cells per testis increases approximately sixfold. The second occurs between 3 and 4 mo of age, or just before puberty, which occurs between 4 to 5 mo of age, when Sertoli cells almost double their numbers per testis. The periods of Sertoli cell proliferation were concomitant with high FSH plasma levels and prominent elongation in the length of seminiferous cord/tubule per testis. Leydig cell volume increased markedly from birth to 1 mo of age and just before puberty. In general, during the first 5 mo after birth, Leydig cell volume growth showed a similar pattern as that observed for testosterone plasma levels. Also, the proliferation of Leydig cells per testis before puberty showed a pattern similar to that observed for Sertoli cells. However, Leydig cell number per testis increased up to 16 mo of age. Substantial changes in Leydig cell size were also observed after the pubertal period. From birth to 4 mo of age, germ cells proliferated continuously, increasing their number approximately two- to fourfold at each monthly interval. A dramatic increase in germ cells per cross-section of seminiferous tubule was observed from 4 to 5 mo of age; their number per tubule cross-section stabilized after 8 mo. To our knowledge, this is the first longitudinal study reporting the pattern of Sertoli cell, germ cell, and Leydig cell proliferative activity in pigs from birth to adulthood and the first study to correlate these events with plasma levels of FSH and testosterone.

We previously reported that rdw rats were infertile in both sexes. The present study was conducted to determine whether hypothyroidism in adult male rdw rats induced infertility by impairing sexual behavior or testicular function, whether the infertility could be reversed by thyroxine (T₄) treatment, and whether the mutant could be produced by infertile rdw rats via in vitro fertilization. The sexual behavior was analyzed by pairing with normal female rats. The fertility of epididymal sperm was determined by in vitro fertilization. The results indicated that the infertility resulted from both defective sexual behavior and testicular function. No untreated rdw rats mated. The weights of epididymides were significantly low, whereas those of testes were not different from those of untreated normal rats. Epididymal sperm with cytoplasmic droplets were observed at a significantly high frequency. No fertilization was detected either in vivo or in vitro. Thyroxine treatment markedly increased serum T₄ levels and the weights of both epididymides and testes. Partial reversion of the impaired sexual behavior was observed, and the percentage of epididymal sperm with cytoplasmic droplets was markedly decreased after T₄ treatment. Fertility of epididymal sperm was completely reversed when determined both in vivo and in vitro, and homozygous embryos developed to term after transfer without loss of viability.

Progesterone (P) is one of several local mediators in the ovulatory cascade in the rat. The precise mechanisms of action for P in ovulation and in what phase of the ovulatory process P is critical, however, need to be clarified. The present study used a selective P-receptor antagonist, Org 31710, in the in vitro perfused rat ovary model to examine the local role of P and possible effects on prostaglandin (PG) and plasminogen-activator (PA) release in ovulation. Ovaries from eCG (15 IU)-primed rats were perfused for 20 h with LH (0.2 μg/ml) and 3-isobutyl-1-methylxanthine (IBMX, 200 μM) to induce ovulation (median = 10.0, 25%–75% range = 8.5–13). Org 31710 was added at either 0, 3.5, 7, or 9 h after LH IBMX, resulting in significant suppression of ovulation after addition at 0 and 3.5 h (1.0, 1–5.5; and 5.0, 2.5–7.75 ovulations, respectively) but no suppressive effect when added at later time points. Progesterone and estradiol levels in the perfusion media were increased after LH IBMX but were not affected by the presence of Org 31710. Ovarian tissue levels of PGE₂, PGF_2α, and PA activity were measured in ovaries that had been perfused for 10 h, a time that was 2 to 5 h before anticipated ovulation. The presence of Org 31710 significantly decreased the levels of PGE₂, PGF_2α, and PA activity. These results suggest that P is essential in ovulation during the initial stages of the ovulatory process. The effect of P to facilitate ovulation seems to relate to stimulation of the PG- and PA-mediator systems.

Results of previous in vitro and in vivo studies have illustrated that the expression of testin by Sertoli cells is tightly associated with the disruption of Sertoli-germ cell junctions. In the present study, treatment of rats with cadmium chloride (CdCl₂), which disrupted the inter-Sertoli tight junctions, failed to induce any changes in testicular testin expression. In contrast, lonidamine, an antispermatogenic drug that rearranges the Sertoli cell membrane microfilament structure causing a disruption of Sertoli-germ cell adhesion junctions, induced a drastic increase in testicular testin expression when administered orally. Lonidamine-induced Sertoli cell testin expression involved both ongoing RNA and de novo protein synthesis. Basal testin expression remained stable during the 27-h incubation with actinomycin D but required de novo protein synthesis in vitro. An inhibitor of protein kinase A, Rp-cAMPS, caused a 50% inhibition of Sertoli cell testin expression at 10 μM within 24 h. A biphasic response was noted in testin expression when forskolin was included in the Sertoli cell culture, and high concentrations of cAMP analogues (1 mM) rapidly reduced testin expression. However, lonidamine can abolish the inhibitory effect of cAMP analogues on Sertoli cell testin expression. These results illustrate that the induction of testin expression may involve several signal transduction pathways.

Follicle-stimulating hormone is the major regulator of growth and development of antral follicles in the ovary. Granulosa cells (GCs) in these follicles are coupled via gap junctions (GJs) consisting of connexin 43 (Cx 43). Because we and others have found that Cx 43 and GJs, respectively, are more abundant in large antral follicles compared with small antral and preantral follicles, we hypothesized that FSH may control Cx 43 gene expression, GJ formation, and intercellular communication. To directly address these points, we chose a rat GC line (GFSHR-17) expressing the FSH receptor and the Cx 43 gene. The functionality of FSH receptors was shown by the effects of porcine FSH, namely cell rounding, reduced cellular proliferation, and stimulation of progesterone production of GFSHR-17 cells, which are effects that were detectable within hours. Treatment with FSH also statistically significantly increased Cx 43 mRNA levels, as shown after 6 to 9 h in Northern blots. These effects were antedated by altered GJ communication, which was observed within seconds. Using a single-cell/whole-cell patch clamp technique, we showed that FSH rapidly and reversibly enhanced electrical cell coupling of GFSHR-17 cells. Increased GJ communication was associated with statistically significantly decreased phosphorylation of Cx 43, which was observed within 10 min after FSH addition, during immunoprecipitation experiments. Our results demonstrate, to our knowledge for the first time, that the gonadotropin FSH acutely and directly stimulates intercellular communication of GFSHR-17 cells through existing GJs. Moreover, FSH also increases levels of Cx 43 mRNA. These changes are associated with reduced proliferation and enhanced differentiation of GFSHR-17 cells. In vivo factors in addition to FSH may be involved in the regulation of GJ/GJ communication between GCs in the follicle, but our results suggest that improved cell-to-cell coupling, enhanced Cx 43 gene expression, and possibly, formation of new GJs are direct consequences of FSH receptor activation and may antedate and/or initiate the pivotal effects of FSH on GCs.

Oocytes secrete factors that regulate the development of the surrounding granulosa cells in ovarian follicles. KIT ligand (KL) mRNA expression in granulosa cells is thought to be regulated by oocytes; however, the factor(s) that mediate this effect are not known. One candidate is the oocyte-specific gene product growth differentiation factor-9 (GDF-9). This study examined the effect of recombinant GDF-9 (rGDF-9) on steady-state KL mRNA expression levels in preantral and mural granulosa cells in vitro. Furthermore, the study compared the effect of rGDF-9 with that of coculture with oocytes at different developmental stages. As determined by RNase protection assay, both KL-1 and KL-2 mRNA levels in preantral and mural granulosa cells were suppressed by 25–250 ng/ml rGDF-9. Fully grown oocytes also suppressed both KL-1 and KL-2 mRNA expression levels. Partly grown oocytes isolated from 7-, 10-, or 12-day-old mice either had no effect on KL mRNA levels or promoted KL-1 mRNA steady-state expression. It is concluded that GDF-9 is likely to mediate the action of fully grown, but not partly grown, oocytes on granulosa cell KL mRNA expression.

In vertebrates, the growth and maturation of the ovarian follicle is dependent on the appropriate dynamics of sex steroid secretion, which is dictated by gene expression of the steroidogenic enzymes. The molecular aspects of steroid regulation are poorly understood in fishes, so as a first step we determined the pattern of expression of four key steroidogenic genes throughout the ovarian cycle in an annually spawning teleost, the channel catfish (Ictalurus punctatus). The abundance of transcripts encoding 3β-hydroxysteroid dehydrogenase (3β-HSD) and cholesterol side chain cleavage (P450_scc), 17α-hydroxylase/lyase (P450_c17), and aromatase (P450_arom) were determined by rtqRT-PCR or ribonuclease protection assay and correlated to ovarian growth and plasma titers of estradiol (E₂) and testosterone (T) in two populations of catfish. Elevations in transcript abundance for P450_c17, P450_scc, and P450_arom were observed at the onset of ovarian recrudescence and during early vitellogenic growth of the oocytes; however, all three decreased precipitously with the completion of vitellogenesis. Changes in the expression of these genes strongly suggest a direct correlation to E₂ and T titers. Alternatively 3β-HSD transcript abundance was relatively stable throughout the year. This study suggests that the genes encoding the three steroidogenic cytochrome P450s have a similar regulatory mechanism.

Programmed cell death occurs spontaneously during spermatogenesis and can be induced in a cell- and stage-specific manner by mild testicular hyperthermia. Studies using transgenic mice suggest the involvement of Bcl-2 proteins in regulating germ cell apoptosis. To delineate further the pathways involved, we examined the temporal changes in proapoptotic Bax and antiapoptotic Bcl-2 in rat testes after transient exposure to heat (43°C for 15 min). Germ cell apoptosis, involving exclusively early (I–IV) and late (XII–XIV) stages, was activated within 6 h. Initiation of apoptosis was preceded by a redistribution of Bax from a cytoplasmic to perinuclear localization within 0.5 h of heating as assessed by immunocytochemical methods. In contrast, Bcl-2 is distributed both in the cytoplasm and nucleus in those cell types susceptible to heat-induced apoptosis. Despite the striking redistribution, Bax levels remained unchanged as determined by Western analysis; Bcl-2 levels increased significantly by 6 h after heat exposure. Reverse transcription-polymerase chain reaction analysis indicated no change in either Bax or Bcl-2 mRNA levels in response to heat, suggesting the involvement of post-transcriptional rather than transcriptional mechanisms mediating their activity. The marked subcellular redistribution of Bax prior to activation of apoptosis and the increase in Bcl-2 suggest an involvement of Bcl-2 family members in heat-induced apoptotic death of germ cells.

Striped bass are seasonal breeding fish, spawning once a year during the spring. All 3-yr-old males are sexually mature; however, 60–64% of the fish mature earlier as 1- or 2-yr-old animals. The endocrine basis underlying early maturity in 2-yr-old males was studied at the molecular level by monitoring changes in pituitary βFSH and βLH mRNA levels by ribonuclease protection assay, and correlating these changes to stages of testicular development. In maturing males, the mRNA levels of βFSH were elevated during early spermatogenesis, whereas βLH mRNA levels peaked during spermiation. The appearance of spermatozoa in the testis was associated with a decrease in βFSH mRNA and a rise in βLH mRNA abundance. Immature males had lower levels of βLH mRNA than maturing males, but there were no differences in βFSH mRNA levels between immature and maturing males. The regulation of gonadotropin gene expression in 2-yr-old males was studied by the chronic administration of GnRH analogue (GnRHa) and testosterone (T), with or without pimozide (P) supplementation. In immature males, the combination of T and GnRHa stimulated a three- to fivefold increase in βFSH and βLH mRNA levels, but the same treatment had no effect on gonadotropin gene expression in maturing males. In addition, the coadministration of P to immature males suppressed the stimulatory effect of GnRHa and T on βFSH and βLH mRNA levels, suggesting that dopamine may have a novel role in regulating gonadotropin gene expression.

POU transcription factors are involved in transcriptional regulation during early embryonic development and cell differentiation. Oct-4, a member of this family, has been shown to be under strict regulation during murine development. The expression of Oct-4 correlates with the undifferentiated cell phenotype of the mouse preimplantation embryo. In this study, expression of a gene construct consisting of selected parts of the region upstream from the murine Oct-4 gene as promoter/enhancer, enhanced green fluorescent protein (EGFP) as reporter and the five exons of the murine Oct-4 gene (GOF18-ΔPE EGFP) was evaluated in murine, porcine, and bovine preimplantation embryos. For comparison, expression of the endogenous Oct-4 gene was also analyzed in all three species by immunocytochemistry. The transgene construct was microinjected into zygotes cultured in vitro to various developmental stages. The EGFP fluorescence was visualized in developing embryos by excitation with blue light at different days following microinjection and showed similar expression patterns in all three species. Most embryos displayed a mosaic pattern of transgene expression. The EGFP fluorescence was not restricted to the inner cell mass (ICM) but was also seen in trophoblastic cells. An affinity-purified polyclonal antibody specific to Oct-4 was used for immunocytochemical analysis of in vivo- and in vitro-derived bovine and porcine blastocysts and also of in vivo-derived murine blastocysts. In the in vivo-derived murine embryos, Oct-4 protein was detectable in the ICM but not the trophectoderm, whereas in porcine and bovine blastocysts, derived in vivo or in vitro, Oct-4 protein was detected in both the ICM and the trophectoderm. Thus, in the two large animal species, Oct-4 expression from the endogenous gene was clearly not restricted to the pluripotent cells of the early embryo. These results show that Oct-4 regulation differs between these species and that the presence of Oct-4 protein may not be sufficient for selection of undifferentiated cell lines in domestic animals.

At fertilization in most animals, cortical granules of the egg or oocyte secrete their contents, whose function it is to modify the extracellular matrix. This modified matrix then participates in the block to polyspermy and protection for early embryonic development. In the sea urchin, contents of the cortical granules are secreted within 30 sec of insemination. Several of these content proteins then bind to the nascent vitelline layer of the egg and lift off the cell surface to form a stable, impervious, fertilization envelope. At least six major proteins are present in the envelope, and recently we have identified cDNA clones of two, ovoperoxidase, and SFE9. Here we report on the identification and characterization of SFE1, a constituent of the fertilization envelope of the sea urchin Strongylocentrotus purpuratus, that has revealing characteristics of how the envelope might form and what protein interaction domains might predominate. We present the largest cDNA sequence we were able to identify representing approximately two thirds of the predicted protein coding region. The C-terminal half of the cognate SFE1 protein contains two different amino acid repeat motifs: a cysteine-rich (15%) motif of 40 amino acids that is tandemly repeated 22 times and is followed by a serine/threonine-rich (38%) repeat of 63 amino acids that is tandemly repeated 3.5 times. Surprisingly, just N-terminal to the cysteine-rich repeat region is a sequence of five repeats with similarity to repeats in another cortical granule protein, SFE9, and to the motif originally identified in the receptor of low-density lipoproteins, the LDLr motif. The amino acid composition deduced from the partial SFE1 cDNA is similar also to the composition of proteoliaisin, a protein thought to tether the ovoperoxidase to the vitelline layer of the egg and thereby sequester the crosslinking activity of the ovoperoxidase to a limited population of proteins in the fertilization envelope. However, by use of monoclonal and polyclonal antibodies to SFE1 and proteoliaisin, we show here that they are distinct gene products. We also show that SFE1 is packed selectively into the cortical granules and then is crosslinked into the fertilization envelope following fertilization. In situ RNA hybridization analysis shows that the mRNA of SFE1 (9 kilobases) is present in oocytes selectively and is turned over rapidly in the oocyte following germinal vesicle breakdown. Our findings suggest that the gene encoding this major product of the egg is activated concomitantly with the other cortical granule-specific products already identified, and that a common LDLr-like motif of the fertilization envelope may reveal a structural mechanism for protein interactions in its construction.

Tenascin-C (TN-C), an extracellular matrix glycoprotein, is known to be expressed in uterine stroma in the peri-implantation period. Examination of the spatiotemporal pattern during early pregnancy using immunohistochemistry and in situ hybridization revealed TN-C expression in the stroma beneath the luminal epithelia of the murine endometrium on Days 0 and 1 of pregnancy, subsequent disappearance, and reappearance on Day 4. After decidualization, tissue around the deciduoma was positive. In situ hybridization demonstrated TN-C production by the stromal cells adjacent to the epithelia. To investigate the regulation of TN-C expression in vitro, murine uterine stromal and epithelial cells were isolated and cultured. Addition of interleukin-1α (IL-1α) and prostaglandin E₂ (PGE₂) and F_2α (PGF_2α) induced TN-C expression in the stromal cells at both protein and mRNA levels, while the sex steroid hormones, progesterone and β-estradiol, exerted little effect. Immunohistochemistry using anti-IL-1α antibody showed epithelial cells to be positive on Days 2–4 of pregnancy, and addition of progesterone but not β-estradiol enhanced IL-1α expression in epithelial cells in vitro. In a culture insert system, TN-C expression by stromal cells cocultured with epithelial cells was induced by addition of progesterone alone that was blocked by additions of anti-IL-1α antibody. Collectively, these findings indicate that TN-C expression in the preimplantation period is under the control of progesterone, but not directly, possibly by the paracrine and autocrine intervention of IL-1α secreted by epithelial cells and PGE₂ and PGF_2α secreted by stromal cells.

Undernutrition has well-established effects on female reproduction. Here we describe the effects of food restriction on aspects of the hypothalamic-pituitary-gonadal (HPG) axis in the female musk shrew. We determined that acute re-feeding reverses deficits brought on by food restriction. Two days of food restriction led to an increase in proGnRH immunoreactive cells in the preoptic area relative to ad libitum-fed controls (AL). This increase was reversed by 90 min of ad libitum feeding in the re-fed females (RF). In addition, food-restricted (FR) females had significantly greater GnRH content in the median eminence than either the AL or RF females. After GnRH was administered, the majority of females in all food conditions ovulated, yet the FR females had significantly fewer corpora lutea than either the AL or RF animals. These data show that food restriction impairs HPG axis function in female musk shrews, and that some of these impairments can be rapidly reversed by acute re-feeding.

In order to evaluate a possible paternal age effect, testicular sperm cells from three men aged 81, 82, and 83 yr were analyzed by two-color- and three-color-fluorescence in situ hybridization for disomy rates of chromosomes 1, 17, 18, X, and Y as well as for diploidy frequencies. A minimum of 1500 sperm cells per donor and probe was evaluated due to the low number of spermatozoa in the preparations. Diploidy and disomy frequencies were in the same range as found in men aged <30 yr, a slight increase only being noticed for XY nuclei.

The present study was conducted to determine the effects of cumulus cells and sodium pyruvate during in vitro maturation of bovine oocytes on maturation, fertilization, and subsequent development. Cumulus-enclosed oocytes (CEOs) and cumulus-denuded oocytes (CDOs) were cultured for 24 h in polyvinylpyrrolidone-Hepes-tissue culture medium 199 with or without sodium pyruvate. Oocytes were fertilized in vitro and then cultured in CR1aa for 10 days. Before in vitro fertilization, the glutathione (GSH) content of some oocytes was measured. Maturation and normal fertilization rates of CDOs cultured with sodium pyruvate and CEOs were higher than that of CDOs cultured without sodium pyruvate. The CEOs showed significantly higher rates of development to the blastocyst stage than CDOs. The GSH contents of oocytes significantly decreased in CDOs after maturation culture, but the GSH contents of oocytes in CEOs remained at the same level as oocytes before culture. These results indicate that sodium pyruvate promotes nuclear maturation of bovine CDOs and that a continuing presence of cumulus cells during maturation is important for subsequent development of zygotes to the blastocyst stage. However, blastocysts produced from CDOs in the presence of sodium pyruvate showed a developmental competence to be normal calves, but it is not known if CDOs cultured without sodium pyruvate also were capable of developing into calves.

Cloning and sequencing of a cDNA amplified by RNA fingerprinting at the implantation site of pregnant rats revealed 80% similarity with Hβ58, previously shown to be essential for formation of the chorioallantoic placenta in the mouse. Hβ58 mRNA was detected in the endometrium of hormonally sensitized rats stimulated to undergo decidualization and in the contralateral uterine horns lacking a decidual stimulus, indicating that uterine expression of Hβ58 mRNA did not require decidualization or the presence of a blastocyst. Immunodetection in the early postimplantation uterus (Days 6–8 of pregnancy) showed Hβ58 localized in the luminal and glandular epithelia and some stromal cells. Decidual cells at Day 6 of pregnancy expressed Hβ58, and by Day 9 of pregnancy, the protein localized throughout the maternal decidua. The temporal and spatial distribution of Hβ58 in the developing chorioallantoic placenta was assessed at Days 10, 12, and 14 of pregnancy. Immunoreactive Hβ58 localized to erythroid cells within the developing fetal vasculature of the chorioallantoic primordia at Day 10 of pregnancy. By Day 12, the fetal vasculature extended into the placental labyrinth, and the erythroid stem cells continued to strongly express Hβ58. At Day 14 of pregnancy, immunoreactivity became evident in the trophoblast giant cells and syncytiotrophoblast of the fetal placenta. As the chorioallantoic placenta matured (Day 18), Hβ58 mRNA was 3.6-fold higher in the labyrinth compared with the junctional region. Stable cell lines (HRP/LRP) isolated from the rat labyrinthine placenta expressed Hβ58 mRNA and protein. The expression pattern of Hβ58 in maternal and fetal placental tissues and its early expression in fetal erythroid stem cells during formation and maturation of the chorioallantoic placenta suggest that Hβ58 plays key roles in the regulatory networks that control hematopoietic development and placentation.

Changes in mRNA expression for estrogen receptor (ERβ) in relation to mRNAs for LH receptor (LHr) and cytochrome P450 enzymes were examined in granulosa and theca cells from proestrous rat ovarian follicles. Of the 30 ovaries harvested from 15 adult rats, 24 were processed for in situ hybridization, and the remaining were used for reverse transcription-polymerase chain reaction. Messenger RNAs for ERβ, LHr, cytochrome P450 side-chain cleavage enzyme (P450_scc), 17α-hydroxylase (P450_c17), aromatase (P450_arom), and steroidogenic acute regulatory protein (StAR) were localized in cross sections of ovaries by in situ hybridization and quantified in granulosa and theca cell layers by a computer-image analyzing system. Ovarian follicles were classified as healthy or atretic. Healthy follicles were divided into four size groups: very small (40–100 μm), small (101–275 μm), medium (276–450 μm), and large (451–850 μm). Atretic follicles were divided into medium (276–450 μm) or large follicles (451–850 μm). A low level of ERβ mRNA expression was first detected in granulosa cells of very small healthy follicles, and the expression increased progressively up to medium-sized follicles. The expression of ERβ mRNA was highest (P < 0.01) in medium-sized follicles that was followed by a decrease (P < 0.01) in large follicles. Messenger RNAs for LHr, P450_scc, and P450_arom were first detected in granulosa cells of medium-sized healthy follicles, while mRNAs for LHr, P450_scc, P450_c17, and StAR were first detected in theca cells associated with very small follicles. The highest expression of LHr, P450_scc, P450_c17, P450_arom, and StAR was seen in granulosa and/or theca cells of large healthy follicles. In atretic follicles, level of gene expression was relatively low in both granulosa and theca cells. In conclusion, stage-specific expression of ERβ mRNA was observed in granulosa cells during follicular development. The increased expression of ERβ and a concomitant initiation of LHr, P450_scc, and P450_arom expression in granulosa cells of medium follicles may signify a role for estrogen in follicular development. Also, a strong correlation between ERβ mRNA expression in granulosa cells, and the expression of mRNAs for LHr, P450_scc, P450_c17, and StAR in theca cells associated with growing follicles suggests a possible role for estrogen in steroidogenesis.

Steroidogenic factor 1 (SF-1) or Ad4BP is a member of the fushi tarazu factor 1 (FTZ-F1) family and an orphan nuclear receptor that plays an important role in the hypothalamus-pituitary-gonadal axis and the adrenal cortex. Although its critical role in the differentiation of adrenals, gonads, and pituitary gonadotropes has been well demonstrated, regulatory function of SF-1 during sexual maturation is yet to be examined. To investigate the potential role of SF-1 in sexual maturation, expression of two salmon FTZ-F1 homolog genes, sFF1-I and sFF1-II, was examined in the pituitaries of chum and sockeye salmons, using specific and sensitive RNase protection assays. Only sFF1-I mRNA was found in the pituitary and other organs, such as the ovary, spleen, liver, brain, and skeletal muscle. In chum salmon during upstream migration from the bay to the hatchery, the level of sFF1-I mRNA in the male fish was increased on the midway in the river, where the levels of gonadotropin α- and IIβ-subunit mRNAs were increased. In maturing sockeye salmon, the expression of the sFF1-I gene was elevated in the mature male fish, but the administration of GnRH analog did not further enhance the expression. These results indicate that sFF1-I gene expression in the pituitary is upregulated in maturing salmon, and this upregulation may not depend on GnRH.

Dynamic regulation of intercellular junctions is an essential aspect of many developmental, reproductive, and physiological processes. We have shown that expression of the desmosomal protein desmoplakin decreases in the luminal uterine epithelium during the preimplantation period of pregnancy in mice. By the time of implantation (between Days 4.5 and 5 of pregnancy), desmoplakin protein can barely be detected by SDS-PAGE and Western blotting, and by immunocytochemistry, it is restricted to well-spaced, punctate dots at the apicolateral junction. Using confocal XZ series and electron microscope quantitation, both the density and distribution of desmosomes along the lateral cell surfaces of luminal epithelial cells were observed to change during early pregnancy. On Day 1 of pregnancy, desmosomes were found at high density in the apicolateral junctional complex, being present here in 79% of ultrathin sections examined, whereas on Day 5, the density was much reduced (present in only 18% of ultrathin sections examined). Desmosomes were found along the lateral surfaces, at or below the level of the nucleus, in 15% of ultrathin sections examined on Day 1 of pregnancy but in only 1% on Day 5. Desmoplakin mRNA declined during the first 4–5 days of pregnancy, along with the protein, suggesting that these changes are controlled at the level of mRNA. This study shows that desmosomes are regulated during early pregnancy, and we propose that a reduction in desmosome adhesion facilitates penetration of the luminal epithelium by trophoblast cells at implantation.

Sperm from C57BL/6J, DBA/2J, BALB/cJ, 129S3/SvImJ, and FVB/NJ inbred mice were cryopreserved in 3% skim milk/18% raffinose cryoprotectant solution. The post-thaw sperm from all strains were evaluated for their viability and fertility by comparing them against B6D2F1 sperm used as a control. The protocol used for freezing mouse sperm was effective in different strains, because the motility was decreased by 50% after cryopreservation similar to other mammalian sperm. However, the progressive motility and the fertility of each inbred strain were affected differently. The C57BL/6J, BALB/cJ, and 129S3/SvImJ strains were the most affected; their fertility (two-cell cleavage) decreased from 70%, 34%, and 84% when using freshly collected sperm to 6%, 12%, and 6% when using frozen/thawed sperm, respectively. Live newborns derived from frozen/thawed sperm were obtained from all strains in the study. These results corroborate the genetic variation among strains with regard to fertility and susceptibility to cryopreservation.

Decidualization is a process characterized by morphological and functional changes in the uterine stromal cells. In addition to steroid hormones, growth factors are implicated in this process. Using in situ hybridization, we found that mRNAs for several bone morphogenetic proteins (BMPs) were detected in the decidual and vascular endothelial cells. The Bmp7 mRNA was detected in the decidualizing stromal cells surrounding the blastocyst and distributed in a gradient, with the highest levels occurring near the uterine epithelium at 4.5 days post-coitus (dpc). With the progression of decidualization, Bmp7 signals in the deciduum at the antimesometrial side decreased, but strong signals were retained in the decidual area at the mesometrial side at 7.0 dpc. In contrast, Bmp8a transcripts increased from 5.5 to 7.0 dpc in the decidual tissue, with the highest levels occurring in the secondary decidual zone at the antimesometrial side. The Bmp2, Bmp4, and Smad1 transcripts were found in the secondary decidual zone, especially at the mesometrial side. The Bmp2 signals were primarily detected in decidual cells, whereas Bmp4 and Smad1 transcripts were mainly detected in vascular endothelial cells, suggesting that they may be involved in decidual angiogenesis.

The production of cloned animals is, at present, an inefficient process. This study focused on the fetal losses that occur between Days 30–90 of gestation. Fetal and placental characteristics were studied from Days 30–90 of gestation using transrectal ultrasonography, maternal pregnancy specific protein b (PSPb) levels, and postslaughter collection of fetal tissue. Pregnancy rates at Day 30 were similar for recipient cows carrying nuclear transfer (NT) and control embryos (45% [54/120] vs. 58% [11/19]), although multiple NT embryos were often transferred into recipients. From Days 30–90, 82% of NT fetuses died, whereas all control pregnancies remained viable. Crown-rump (CR) length was less in those fetuses that were destined to die before Day 90, but no significant difference was found between the CR lengths of NT and control fetuses that survived to Day 90. Maternal PSPb levels at Days 30 and 50 of gestation were not predictive of fetal survival to Day 90. The placentas of six cloned and four control (in vivo or in vitro fertilized) bovine pregnancies were compared between Days 35 and 60 of gestation. Two cloned placentas showed rudimentary development, as indicated by flat, cuboidal trophoblastic epithelium and reduced vascularization, whereas two others possessed a reduced number of barely discernable cotyledonary areas. The remaining two cloned placentas were similar to the controls, although one contained hemorrhagic cotyledons. Poor viability of cloned fetuses during Days 35–60 was associated with either rudimentary or marginal chorioallantoic development. Our findings suggest that future research should focus on factors that promote placental and vascular growth and on fetomaternal interactions that promote placental attachment and villous formation.

The effects of retinoic acid on the development of reproductive organs and egg production in female Japanese quail (Coturnix coturnix japonica) were investigated. Female quail were fed a diet containing retinoic acid at 4 mg/kg (RA) or two diets containing retinyl acetate at 5000 IU/kg (VA1) or 14 000 IU/kg (VA2) after being fed a vitamin A-free diet for 2 wk (experiment 1). The oviduct and ovary grew more rapidly (P < 0.05) in RA-treated quail than in VA-treated quail at 5 wk of age. In addition, the body weight of RA-fed quail was also greater (P < 0.05) than that of VA-fed quail at 5 wk. The RA-treated quail laid their first eggs approximately 5 days earlier (P < 0.05) than the VA-treated quail. Furthermore, these RA-fed quail laid more eggs (P < 0.05) than those VA-fed quail during the experimental period. To confirm the results of experiment 1, a similar experiment was conducted to record the first egg and total eggs laid by quail fed VA2 or RA (experiment 2). The early onset of oviposition was again observed in the RA-treated group (P < 0.01). These results suggest that retinoic acid has a stimulating effect on the reproductive system of female Japanese quail, as has been previously shown in the reproductive system of male Japanese quail.

The nucleus of mammalian spermatozoa is surrounded by a rigid layer, the perinuclear theca, which is divided into a subacrosomal layer and a postacrosomal calyx. Among the proteins characterized in the perinuclear theca, calicin is one of the main components of the calyx. Its sequence contains three kelch repeats and a BTB/POZ domain. We have studied the association of boar calicin with F-actin and the distribution of boar and human calicin during spermiogenesis compared with the distribution of actin. Calicin was purified from boar sperm heads under nondenaturating conditions. The molecule bound actin with high affinity (K_d = ∼5 nM), and a stoichiometry of approximately one calicin per 12 actin monomers was observed. Gel filtration studies showed that calicin forms homomultimers (tetramers and higher polymers). According to immunocytochemical results, calicin is present (together with actin) in the acrosomal region of round spermatids and is mainly localized in the postacrosomal region of late spermatids and spermatozoa. Taken together, the results suggest that the affinity of calicin to F-actin allows targeting of calicin at the subacrosomal space of round spermatids, and that its ability to form homomultimers contributes to the formation of a rigid calyx.

Apoptosis, or programmed cell death, is an important mechanism for the regulation of embryonic development and tissue homeostasis. It is coordinated by a number of molecules including the Fas-Fas ligand (FasL) system and bcl-2. The purpose of this study was to characterize the expression of these molecules in human oocytes and cumulus cells from gonadotropin-stimulated human ovaries and to determine whether the presence of soluble Fas (sFas), soluble FasL, or interferon-γ in follicular fluid (FF) correlated with apoptosis in cumulus cells, oocyte maturation, and embryo quality. Levels of sFas were significantly higher in FF containing immature oocytes compared with those containing atretic oocytes (P < 0.05; FF containing mature oocytes had highly variable levels of sFas. Levels of sFas in FF did not correlate with either fertilization, embryo quality resulting from fertilized oocytes, or apoptosis rate in cumulus cells. Fas was expressed in both unfertilized oocytes and cumulus cells, whereas FasL expression was not usually detected in these cell types. Messenger RNA for bcl-2 was detectable in both freshly isolated oocytes and cumulus cells but was not demonstrable following 24 h of culture that coincided with a significant increase of apoptosis in cumulus cells. Our results indicate that soluble forms of the Fas-FasL system are present in FF from gonadotropin-stimulated human ovaries and suggest that this system may play a role in preventing oocyte atresia during folliculogenesis but is probably not important for apoptotic events in cumulus cells and oocytes after fertilizaton failure. Apoptosis in this case may be facilitated by the downregulation of bcl-2. Further studies on the expression of these molecules in follicles containing atretic oocytes and immature oocytes are needed to confirm this new hypothesis.

It is well known that the transfer of immunoglobulins (Igs) from mother to young via milk contributes to the offspring's immune defense. The present study suggests that not only is IgG transmitted to progeny, but that functional maternal Ig-secreting cells (or B cells) can also be transferred to the neonate. We have used B cell-deficient (μ^−/−) mice and found that a high proportion of them obtain long-lasting, partial reconstitution of their serum Ig levels if born to μ ^/− mothers. In some of these serum IgG-positive μ^−/− mice, Ig-secreting cells were detected in spleen and bone marrow. To ensure that cells of maternal origin were present in the progeny, μ^−/− offspring born to μ ^/− dams transgenic for green fluorescent protein (GFP) were used. In spleens and bone marrow from some of these μ^−/−GFP^−/− offspring, GFP-positive cells were detected, which demonstrated that cells of maternal origin could infiltrate the progeny. In addition, splenic Ig-secreting cells were detected in μ^−/− mice that were born to μ^−/− dams and transferred to a lactating μ ^/ foster dam at birth. This indicates that maternal Ig-secreting cells can be transferred postnatally via milk.

Testes from adult and prepubertal mice lacking the Desert hedgehog (Dhh) gene were examined in order to describe further the role of Dhh in spermatogenesis because, in a previous report, Dhh-null male mice were shown to be sterile. Dhh is a signaling molecule expressed by Sertoli cells. Its receptor, patched (Ptc), has been previously localized to Leydig cells and is herein described as being localized also to peritubular cells. Two phenotypes of the mice were observed: masculinized (7.5% of Dhh-null males) and feminized (92.5%), both of which displayed abnormal peritubular tissue and severely restricted spermatogenesis. Testes from adult feminized animals lacked adult-type Leydig cells and displayed numerous undifferentiated fibroblastic cells in the interstitium that produced abundant collagen. The basal lamina, normally present between the myoid cells and Sertoli cells, was focally absent. We speculate that the abnormal basal lamina contributed to other characteristics, such as extracordal gonocytes, apolar Sertoli cells, and anastomotic seminiferous tubules. The two Dhh-null phenotypes described have common peritubular cell defects that may be indicative of the essential role of peritubular cells in development of tubular morphology, the differentiation of Leydig cells, and the ultimate support of spermatogenesis.

To establish a systematic strategy for characterizing fertilization proteins of sperm cells, we prepared alloantisera by immunizing gilts with salt-washed membranes from boar spermatozoa. The antisera recognized a unique subset of sperm membrane proteins that migrated with M_r 7500–66 000 in SDS-PAGE under nonreducing conditions. The antisera did not recognize proteins of erythrocyte membranes, and tissue absorption experiments further confirmed that the alloantigens were sperm-specific proteins. Each of these sperm-specific membrane proteins (SSMPs) possessed one or more disulfide bonds that were essential for its interaction with alloantibody. Enzymatic deglycosylation revealed that most of the SSMPs were glycoproteins, and their alloantigenicity was not dependent on the presence of N-linked oligosaccharides. The presence of disulfide bonds and glycosylation indicated that the SSMPs identified each comprise at least one extracellular domain. Two-dimensional electrophoresis resolved at least 14 distinct SSMPs, 13 of which possessed acidic pIs (range 4.2–4.8). By indirect immunofluorescence, the SSMPs localized to the cell surface overlying all major regions of the sperm cell. We conclude that the repertoire of immunodominant SSMPs in the pig is relatively small, which makes feasible the systematic elucidation of their functions in fertilization.

Ribosomal RNA genes are transcribed in the nucleolus. The formation of this organelle after fertilization is essential for embryonic protein synthesis and viability. We have examined nucleolus formation in in vivo-derived porcine embryos by light microscopical autoradiography following 20 min of ³H-uridine incubation, transmission electron microscopy (TEM), and immunocytochemical localization by confocal laser scanning microscopy of key nucleolar proteins involved in rRNA transcription (nucleolin, upstream binding factor, topoisomerase I, and RNA polymerase I) and processing (fibrillarin, nucleophosmin). During the first two postfertilization cell cycles, TEM revealed fibrillar spheres as the most prominent intranuclear entity of the blastomeres. Fibrillogranular nucleoli were established during the third cell cycle. Initially, fibrillar centers, a dense fibrillar component, and a granular component were formed on the surface of the fibrillar spheres. At the same time, autoradiographic labeling over the nucleoplasm and in particular the nucleoli was detected for the first time. The nucleolar proteins were, in general, not immunocytochemically localized to the presumptive nucleolar compartment until late during the third or early during the fourth cell cycle.

A full-length cDNA encoding a GnRH receptor (GnRH-R) has been obtained from the brain of rainbow trout. This cDNA encodes a protein of 386 amino acids (aa) exhibiting the typical arrangement of the G-protein-coupled receptors in seven transmembrane domains. However, a second ATG could give rise to a receptor with a 30-aa longer extracellular domain. As already shown in other fish and Xenopus, this protein possesses an intracellular domain, in contrast with its mammalian counterparts. In the case of rainbow trout, this intracellular carboxy-terminal tail consists of 58 residues. Northern blotting experiments carried out in the brain, the pituitary, and the liver only resulted in a single band of 1.9–2 kilobases in the pituitary, although reverse transcription-polymerase chain reaction amplification products were found in the brain, the pituitary, the retina, and the ovary. In situ hybridization using a probe corresponding to the full-length coding region of the receptor was performed on vitellogenic or ovulating females and allowed to detect a weak but specific signal in the proximal pars distalis of the pituitary, the preoptic region, the mediobasal hypothalamus, and the optic tectum. However, the strongest signal was consistently detected in a mesencephalic structure, the nucleus lateralis valvulae, the significance of which is presently open to speculation.

Silefrin is a sodefrin-like, female-attracting pheromone comprising 10 amino acids that was isolated from the abdominal gland of the sword-tailed newt, Cynops ensicauda. Hormonal effects on the silefrin precursor mRNA expression and silefrin content in the abdominal gland were investigated in the present study by using Northern blot analysis and radioimmunoassay, respectively. In the abdominal gland of newts treated with prolactin (PRL) plus testosterone propionate (TP), silefrin precursor mRNA expression was markedly enhanced as compared with that in the newts injected with saline, PRL, or TP. Values for radioimmunoassayable silefrin content in the abdominal gland paralleled those for the silefrin precursor mRNA levels. Moreover, silefrin precursor mRNA signals, as revealed by in situ hybridization, as well as stainability of immunoreactive silefrin were much more intense in the epithelial cells of the abdominal gland of the PRL-plus-TP-treated animals than in those of controls. We thus conclude that PRL and androgen are important factors for enhancing silefrin synthesis.

Previous studies of the estrogen receptor-alpha knockout (αERKO) in the male mouse demonstrate that the rete testis and efferent ductules are targets of estrogen. Because the αERKO mouse lacks a functional estrogen receptor alpha (ERα) throughout development, it was not known whether the morphological and physiological abnormalities observed in the αERKO male were due to developmental defects or to dysfunctions concurrent with the lack of ERα in the tissue. This study was designed to determine if treatment of normal wild-type (WT) mice with the pure antiestrogen, ICI 182,780, (ICI) could reproduce the morphological characteristics seen in αERKO mice. Thirty-day-old male mice were treated for 35 days with either castor oil or ICI. Age-equivalent αERKO mice were used for comparison. Light microscopic examinations of the reproductive tracts revealed dramatic changes in the efferent ductules of treated mice: a 1.7-fold increase in luminal diameter, a 56% reduction in epithelial cell height, a 60% reduction in brush boarder height of nonciliated cells, and an apparent reduction of the number of observable lysosomes and endocytotic vesicles. Testes of ICI-treated mice showed swollen rete testes area (6.5 times larger than control) and a 65% reduction in rete testis epithelium height. However, there were no significant changes in body and testis weights. These results indicate that ER blockage with ICI in WT mice results in morphological changes of the efferent ductules resembling those seen in αERKO siblings of the same age. Based on this study, we conclude that ERα has a functional role in the mouse reproductive tract and the aberrant morphology observed in the efferent ductules of the αERKO mouse is likely the result of a concurrent response to the lack of functional ERα, and not solely due to the lack of ERα during early developmental times.

The epididymis provides the optimal milieu for sperm maturation and storage. Epididymal secretory proteins are believed to be involved in that process. Androgens are the major endocrine and paracrine regulatory signals that regulate gene expression in the epididymis. We have previously identified an androgen-dependent retinoic acid-binding protein (mE-RABP) that is secreted into the luminal fluid from the mouse mid/distal caput epididymidis. The mE-RABP protein belongs to the lipocalin superfamily and may be involved in the trafficking of retinoic acid within the epididymis. We have recently demonstrated that 5 kilobases of the 5′ flanking region of the mE-RABP gene contained all the information for the hormonal regulation and the tissue-, region-, and cell-specific expression of the mE-RABP gene. In this study, we have identified a complex androgen-specific response region (ARR) within the first 600 base pairs of the mE-RABP gene promoter. Androgen (DHT) but not glucocorticoid (DEX) activates the ARR in HeLa and PC-3 cells. Two androgen receptor binding sites have been located at positions −445/−459 and −102/−88 and were named ARBS-1 and ARBS-0, respectively. Point mutations of ARBS-0 resulted in a slight decrease of the androgen response. However, mutations of ARBS-1 led to a total loss of the androgen responsiveness, suggesting that it was a major cis-acting element. When ARBS-1 is isolated from its promoter context, it serves as a weak androgen-responsive element that was activated by both androgens and glucocorticoids. Also, the −543/−88 DNA promoter fragment behaved as a poor androgen-responsive region, suggesting that regulatory elements located within the proximal mE-RABP promoter were required for a full androgen response. In conclusion, the mE-RABP ARR is a good model for the study of molecular mechanisms that lead to an androgen-specific responsiveness in vivo.

Using immunohistochemistry, the expression of the D-type cyclin proteins was studied in the developing and adult mouse testis. Both during testicular development and in adult testis, cyclin D₁ is expressed only in proliferating gonocytes and spermatogonia, indicating a role for cyclin D₁ in spermatogonial proliferation, in particular during the G₁/S phase transition. Cyclin D₂ is first expressed at the start of spermatogenesis when gonocytes produce A₁ spermatogonia. In the adult testis, cyclin D₂ is expressed in spermatogonia around stage VIII of the seminiferous epithelium when A_al spermatogonia differentiate into A₁ spermatogonia and also in spermatocytes and spermatids. To further elucidate the role of cyclin D₂ during spermatogenesis, cyclin D₂ expression was studied in vitamin A-deficient testis. Cyclin D₂ was not expressed in the undifferentiated A spermatogonia in vitamin A-deficient testis but was strongly induced in these cells after the induction of differentiation of most of these cells into A₁ spermatogonia by administration of retinoic acid. Overall, cyclin D₂ seems to play a role at the crucial differentiation step of undifferentiated spermatogonia into A₁ spermatogonia. Cyclin D₃ is expressed in both proliferating and quiescent gonocytes during testis development. Cyclin D₃ expression was found in terminally differentiated Sertoli cells, in Leydig cells, and in spermatogonia in adult testis. Hence, although cyclin D₃ may control G₁/S transition in spermatogonia, it probably has a different role in Sertoli and Leydig cells. In conclusion, the three D-type cyclins are differentially expressed during spermatogenesis. In spermatogonia, cyclins D₁ and D₃ seem to be involved in cell cycle regulation, whereas cyclin D₂ likely has a role in spermatogonial differentiation.

Term and preterm labor are associated with increased fetal hypothalamic-pituitary-adrenal (HPA) activation and synthesis of prostaglandins (PGs) generated through the increased expression of prostaglandin H synthase-II (PGHS-II) in the placenta. Inhibition of PGHS-II has been advocated as a means of producing uterine tocolysis, but the effects of such treatment on fetal endocrine functions have not been thoroughly examined. Because PGE₂ is known to activate the fetal HPA axis, we hypothesized that administration of meloxicam, a PGHS-II inhibitor, to sheep in induced labor would suppress fetal HPA function. Chronically catheterized pregnant ewes were treated with RU486, a progesterone receptor antagonist, to produce active labor, and then treated with either high-maintenance-dose meloxicam, graded-maintenance-dose meloxicam, or a saline infusion. Maternal uterine contraction frequency increased 24 h after the RU486 injection and the animals were in active labor by 48 ± 4 h. RU486 injection led to increased concentrations of PGE₂, ACTH, and cortisol in the fetal circulation, and increased concentrations of 13,14 dihydro 15-ketoprostaglandin F_2α (PGFM) in the maternal circulation. Uterine activity was inhibited within 12 h of beginning meloxicam infusion at both infusion regimes. During meloxicam infusion there were significant decreases in fetal plasma PGE₂, ACTH, and cortisol concentrations, and PGFM concentrations in maternal plasma. In control animals, frequency of uterine contractions, maternal plasma PGFM, fetal plasma PGE₂, ACTH, and cortisol concentrations increased after RU486 administration, and continued to rise during saline infusion until delivery occurred. We conclude that RU486-provoked labor in sheep is associated with activation of fetal HPA function, and that this is attenuated during meloxicam treatment to a level considered compatible with pregnancy maintenance.

The role of tumor necrosis factor α (TNFα) and its type I receptor (TNFRI) in structural luteolysis was investigated. A semiquatitative reverse-transcription polymerase chain reaction (RT-PCR) was used to characterize the pattern of TNFRI mRNA expression within the corpus luteum (CL) throughout the estrous cycle and its cellular distribution. Increase in TNFRI mRNA levels was recorded both in regressed luteal tissue and in CL of cows injected with prostaglandin F_2α. All three major cell types composing the CL, steroidogenic (large and small) and endothelial cells expressed the TNFRI gene. A densitometric analysis of TNFRI mRNA expression revealed that resident endothelial cells had significantly higher levels of TNFRI mRNA than steroidogenic luteal cells. The physiological effects associated with TNFRI expression were investigated in the various luteal cell types. TNFα-induced programmed cell death (PCD) in dose- and time-dependent manners of cultured luteal endothelial cells (LECs) but not of in vitro luteinized steroidogenic cells. Several lines of evidence are provided to show that progesterone regulates luteal cell survival: 1) CL and LECs express progesterone receptor mRNA, 2) physiological levels of the steroid abolished TNFα-induced PCD of LECs, and 3) progesterone-producing cells are protected from PCD. In conclusion, this study suggests that TNFα-induced PCD during structural luteolysis is mediated by TNFRI, primarily affects endothelial cells, and that the decline in progesterone, preceding structural luteolysis, is a prerequisite for the initiation of apoptosis in endothelial cells.

Apoptosis, or physiological cell death, is elevated in the placenta of human pregnancies complicated by fetal growth retardation, suggesting that placental apoptosis may be a key factor in the overall control of feto-placental growth. The present study used DNA internucleosomal fragmentation analysis to characterize apoptosis in the two morphologically and functionally distinct regions of the rat placenta, the basal and labyrinth zones, during the last week of pregnancy (Days 16, 22, and 23). In addition, because glucocorticoids are potent inhibitors of feto-placental growth and can stimulate apoptosis in other tissues, we examined whether dexamethasone treatment in vivo induces placental apoptosis. DNA fragmentation was clearly evident in both placental zones at each stage of pregnancy, with higher levels evident in the basal zone compared with the labyrinth zone on Days 22 and 23. TUNEL analysis, which identifies dying cells in situ, demonstrated positive staining of cells in the basal zone, particularly giant trophoblast cells. Dexamethasone treatment increased DNA fragmentation in the basal zone but not the labyrinth zone. Similarly, maternal treatment with carbenoxolone, which can enhance local concentrations of endogenous glucocorticoid by inhibition of 11β-hydroxysteroid dehydrogenase, also increased DNA fragmentation in the basal zone but not in the labyrinth zone. These effects of dexamethasone and carbenoxolone on placental apoptosis were associated with reduced placental and fetal weights. In conclusion, this study shows that apoptosis occurs in both zones of the rat placenta, particularly in the basal zone near term, and is elevated after increased glucocorticoid exposure in vivo. These data support the hypothesis that placental apoptosis is an important player in the regulation of feto-placental growth, and establish the rat as a useful model to study the endocrine control of placental apoptosis.

The present study investigated the inhibitory effect of extracellular ATP on Na absorption and the possible underlying mechanism in cultured mouse endometrial epithelium using the short-circuit current (I_SC) technique. The cultured epithelia exhibited a Na -dependent basal current that could be predominately blocked by the epithelial Na channel (ENaC) blocker, amiloride (10 μM). Apical addition of ATP (10 μM) induced a reduction in basal I_SC. However, in the presence of amiloride or when apical Na was removed, the ATP-induced reduction was abolished and an increase in the I_SC was observed with kinetic characteristics similar to those reported previously for the ATP-induced Cl⁻ secretion, indicating that ATP could induce both Cl⁻ secretion and inhibition of Na absorption. Further reduction in I_SC after ATP challenge could be obtained with forskolin (10 μM), which indicates that different inhibitory mechanisms are involved. The ATP-induced inhibition of Na absorption, but not that induced by forskolin, could be abolished by the P₂ receptor antagonist, reactive blue (100 μM), indicating the involvement of a P₂ receptor in mediating the ATP response. ATP and uridine 5′-diphosphate (UDP; 100 μM), a relatively selective agonist for the pyrimidinoceptor, induced separate I_SC reduction, and distinct I_SC increases in the presence of amiloride, regardless of the order of drug administration, indicating the involvement of two receptor populations. The ATP-induced inhibition of Na absorption was mimicked by the Ca² ionophore, ionomycin (1 μM), whereas the Ca² chelators, EGTA and BAPTA-AM, abolished the ATP-induced, but not the forskolin-induced, inhibition of Na absorption, suggesting the involvement of a Ca² -dependent pathway. In the presence of the Cl⁻ channel blocker, DIDS (100 μM), both inhibitory and stimulatory responses to ATP were abolished, suggesting the involvement of a Ca² -activated Cl⁻ channels (CaCCs) in mediating both ATP responses. The ATP-induced as well as the forskolin-induced reduction in I_SC was not observed when Cl⁻ was removed from the bathing solution, indicating that Cl⁻ permeation is important for the inhibition of Na absorption. The results suggest the presence of a Ca² -dependent ENaC-inhibiting mechanism involving CaCC in mouse endometrial epithelial cells. Thus, extracellular nucleotides may play an important role in the fine-tuning of the uterine fluid microenvironment by regulating both Cl⁻ secretion and Na absorption across the endometrium.