In this review I want to argue that, far from being a macho entity with an all-powerful role in male development, the human Y chromosome is a “wimp.” It is merely a relic of the X chromosome, and most or all of the genes it bears—including the genes that determine sex and control spermatogenesis—are relics of genes on the X chromosome that have other functions altogether.

Blastomeres from eight-cell-stage rabbit embryos have been fused with enucleated metaphase II oocytes (ooplasts) or with ooplasts that were preactivated before fusion. Preactivation of ooplasts before nuclear transfer (NT) raises the rate of preimplantation development from 15% to 56%, which remains elevated in the next series of NT (48.6% and 47.2% in the second and third rounds, respectively). Transfer of eight-cell embryos from the third round to the recipient resulted in the birth of normal young. Synchronization of blastomere nuclei in the G1 phase with nocodazole before fusion results in 42% morula/blastocyst formation. However, in the second generation of NT embryos, the yield drops to as low as 17%, indicating deleterious effects of the second nocodazole treatment on blastomeres. The calculated number of clones per one round of cloning was 4.5, 3.9, and 3.8 in subsequent series; the highest number of morulae and blastocysts that developed from individual donor embryos after three rounds were 26 and 27, respectively.

A large number of natural killer (NK) cells appear in human uterine mucosa during the secretory phase and first trimester pregnancy. We investigated the expression of interleukin (IL)-15, a possible stimulator for these NK cells, in human endometrium and first trimester decidua. Semiquantitative reverse transcriptase-polymerase chain reaction revealed that IL-15 mRNA expression was stronger during the secretory phase and first trimester pregnancy than during the proliferative phase. Immunohistochemistry revealed that immunoreactivity for anti-IL-15 was higher during the secretory phase than it was during the proliferative phase. This was prominent in the perivascular stromal cells around invading spiral arteries during the mid- to late-secretory phase. In first trimester decidua, endothelial cells were also stained as strongly as stromal cells. A membrane-bound IL-15 molecule was detected on the surface of first trimester decidual cells by flow cytometry. Progesterone stimulated the release of soluble IL-15 in the supernatant of cultured decidual cells. These results suggest that IL-15 expression in human uterine mucosa corresponds to the fluctuation of uterine NK cells and that its production is hormonally controlled, especially by progesterone.

During spermatogenesis, spermatids synthesize constituent proteins present in mature spermatozoa; however, little information exists on the molecular processes involved. In previous studies, this laboratory reported the characterization of rat sperm β-d-galactosidase. In this paper, we report the localization of this enzyme along with its biosynthesis and processing. An antibody against rat luminal fluid β-d-galactosidase was used to immunolocalize the enzyme in the testis and in epididymal spermatozoa. We found that β-d-galactosidase is localized within the acrosomal cap of spermatids and in the acrosome and cytoplasmic droplet of epididymal spermatozoa. A combination of germ cell radiolabeling, immunoprecipitation, SDS-PAGE, and autoradiography revealed that spermatids produce two forms of β-d-galactosidase, 90 and 88 kDa. During pulse-chase analysis, a 56-kDa form appeared. Treatment of β-d-galactosidase immunoprecipitates from testicular spermatozoa with N-glycanase or Endo H revealed that both the 90- and 88-kDa forms become a 70-kDa polypeptide on SDS-PAGE. Since Endo H or N-glycanase treatment provided similar results, the presence of extensive N-linked high mannose/hybrid-type glycans on these proteins is indicated. Treatment of the 56-kDa form of β-d-galactosidase with Endo H or N-glycanase resulted in the appearance of 52- and 50-kDa forms, respectively. This result suggests that the 56-kDa form contains N-linked high mannose/hybrid as well as complex oligosaccharides. During epididymal maturation, the 90-kDa form of β-d-galactosidase persists in caput epididymal spermatozoa and is gradually converted to a major 74-kDa form in cauda spermatozoa. In addition to the 90- to 74-kDa forms, cauda spermatozoa show a 56- to 52-kDa form on Western immunoblots. Since only the high-molecular weight forms of β-d-galactosidase are present on immunoblots of isolated sperm heads, we suggest that they are acrosomal in origin and that the 56-kDa form, which is processed to 52 kDa in cauda spermatozoa, is associated with the cytoplasmic droplet.

The relaxin knockout (rlx −/−) mouse was used to assess the effect, during pregnancy, of relaxin with regard to water, collagen content, growth, and morphology of the nipple (N), vagina (V), uterus, cervix (C), pubic symphysis (PS), and mammary gland (MG). The results presented here indicate that during pregnancy, relaxin increases the growth of the N, C, V, and PS. Large increases in water content in the PS (20%) occurred in pregnant (Day 18.5) wild-type (rlx / ) mice but not in rlx −/− animals. This indicates that in the PS, relaxin might increase the concentration of a water-retaining extracellular matrix component (hyaluronate). In the pregnant rlx / mouse, collagen content decreased significantly in the N and V but not in other tissues. There were no significant changes in the rlx −/− mouse. This contrasts with findings in the rat, in which relaxin has been found to cause decreases in collagen concentrations in the V, C, and PS. Histological analysis showed that the collagen stain was more condensed in the tissues (V, C, PS, N, and MG) of rlx −/− mice than in those of rlx / mice. This phenomenon indicates that the failure of collagen degradation and lack of growth in the N underlie the inability of the rlx −/− mice to feed their young, as reported previously. Vaginal and cervical luminal epithelia, which proliferated markedly in the rlx / pregnant mice, remained relatively atrophic in the rlx −/− mice. As proliferation and differentiation of uterine and vaginal epithelia are thought to be induced by a paracrine stromal factor that acts upon estrogen stimulation, our results indicate that relaxin may be this paracrine factor.

Penile erection is mediated by nitric oxide (NO) synthesized by the neuronal nitric oxide synthase (nNOS). In the rat penis, the main nNOS mRNA variant, PnNOS, differs from cerebellar nNOS (CnNOS) by a 102 base pair insert encoding a 34-amino acid sequence. In the mouse, two nNOS mRNAs have been identified: nNOSα, encoding a 155-kDa protein, and an exon 2-deletion variant, nNOSß, encoding a 135-kDa protein that lacks a domain where a protein inhibitor of nNOS (PIN) binds. We wished to determine whether PnNOSα and β are expressed in the rat penis and are located in the nerves and whether the β form persists in the potent nNOS knock-out mouse (nNOS^△△). A PnNOS antibody against the insert common to both PnNOSα and β detected the expected 155-kDa protein in PnNOSα-transfected cells. This antibody, and the one common to PnNOS/CnNOS, showed (on Western blots) the 155- and 135-kDa nNOS variants in rat penile tissue during development and aging. PnNOSα mRNA and its subvariants were found as the main nNOS in the penile corpora, the cavernosal nerve, and the pelvic ganglia, with lower levels of PnNOSβ mRNA. In tissue sections, PnNOS protein was immunodetected in the penile nerve endings in the rat and in the nNOS wild-type and nNOS^△△ mice. An antibody against the sequence encoded by exon 2 did not react (on Western blots) with the 135-kDa band, which confirms that this protein is the β form. In conclusion, both PnNOSα and β are expressed in the rat penis at all ages and are located in the nerves. The β form may allow nitric oxide synthesis during erection to be partially insensitive to PIN. The residual expression of PnNOS, and possibly CnNOS, in the penis of the nNOS^△△ mouse occurs through transcription of the β mRNA, and this may explain the retention of erectile function when the expression of nNOSα is disrupted.

Deterioration in the quality of mammalian oocytes during the metaphase-II arrest period is well known as “oocyte aging.” Oocytes in which aging has occurred are called aged oocytes, and these oocytes show enhanced activation and higher fragmentation rates after parthenogenetic activation. Previously we showed that porcine aged oocytes had low maturation/M-phase promoting factor (MPF) activity, and we suggested that this low MPF activity contributed at least in part to the aging phenomena. In the present study, we examined the relationship between MPF activity and these aging phenomena by artificially regulating MPF activity in porcine metaphase-II-arrested oocytes. Since we have shown recently that aged porcine oocytes contain abundant phosphorylated inactive MPF, so-called pre-MPF, we used vanadate and caffeine, which affect the phosphorylation status of MPF, to regulate MPF activity. Incubation of 48-h-matured oocytes with vanadate for 1 h increased the phosphorylation of MPF and decreased MPF activity. The parthenogenetic activation and fragmentation rates were significantly increased compared with those of control oocytes. Conversely, treatment of 72-h-cultured aged oocytes with caffeine (last 10 h of culture) decreased the level of pre-MPF and elevated MPF activity. These oocytes revealed significantly lower parthenogenetic activation rates and a lower percentage of fragmentation than did untreated aged oocytes. These results indicate that not only the increased ability for parthenogenetic activation but also the increased fragmentation rate observed in porcine aged oocytes may be attributable in part to the gradual decrease in MPF activity during prolonged culture. Control of MPF phosphorylation with these agents may allow for some degree of manipulation of oocyte aging.

Oocyte maturation is a key issue of current animal biotechnology. This study was designed to examine the morphodynamics of the cumulus-oocyte association during oocyte maturation. Porcine cumulus-oocyte complexes were recovered from slaughterhouse ovaries; matured in vitro for 0, 24, 36, and 44 h; and evaluated by scanning electron microscopy either combined or not combined with the osmium-dimethyl sulfoxide-osmium maceration (ODO) method. The cytoskeleton distribution was also observed by fluorescence staining. Prior to maturation culture (0 h), the spherical cumulus cells were tightly clustered around the oocyte, with narrow intercellular spaces. They showed active secretion at 36 h and were fully expanded at 44 h of culture. The ODO methods revealed that the cumulus cells projected numerous long and thin transzonal projections at 0 h, but these were largely disconnected at 44 h. The outer surface of the zona pellucida showed a meshwork surface regardless of time of incubation, whereas the inner surface changed from a fine fibrous surface to a spongy surface that was coated with mucin. The vitelline surface changed from a sparse distribution of short microvilli (MV) to a dense distribution of well-developed MV. Fluorescence staining showed that the cumulus cell projections consisted mainly of microfilaments, which were abundant at the germinal vesicle and metaphase-I (M-I) stages (0–24 h) but which were decreased in number at the M-II stage (36–44 h). We conclude that the cumulus-oocyte transzonal projections became disconnected between the M-I and M-II stages as a result of cumulus expansion. The cumulus-cumulus communications, however, remained intact at these stages, although the biological functions of these communications were not clear.

The detrimental effects of spinal cord injury (SCI) on spermatogenesis in the rat can be attenuated by exogenous testosterone (T) but enhanced by exogenous follicle-stimulating hormone (FSH). These results suggest that T-dependent cellular events may be involved in testicular injury after SCI and that such events may be associated with modification of FSH effects on Sertoli cell function. The current study compared the responses of Sertoli cells to exogenous T and FSH after SCI or sham surgery using steady-state levels of Sertoli cell protein mRNA transcripts as markers of responsiveness. Rats underwent sham surgery or SCI and then were treated for 7 or 14 days with T-filled silastic capsules (2 × 5 cm) and/or daily injections of 0.1 units of porcine FSH. Vehicle-treated control rats received 5-cm empty capsules and daily injections of saline vehicle. Two weeks after sham surgery, levels of mRNA for the androgen receptor (AR), FSH receptor (FSHR), androgen-binding protein (ABP), or sulfated glycoprotein (SGP)-2 in the testis were unaffected by T or FSH alone. Testosterone alone, however, significantly decreased transferrin (Trf) mRNA levels in the testis (P < 0.01). The combination of T and FSH treatments resulted in significant decreases in levels of the above transcripts (P < 0.05; P < 0.01). Seven days after SCI, the testes of vehicle-treated SCI rats had higher levels of AR and SGP-2 mRNA than did those of sham control rats (P < 0.01); such effects were transient and disappeared by Day 14 post-SCI. Testosterone treatment of SCI rats for 7 days resulted in decreases in mRNA levels for AR and Trf in the testes (P < 0.01) but increased testicular levels of mRNAs for FSHR and SGP-2 in SCI rats. Follicle-stimulating hormone treatment for 7 days prevented the increase in AR mRNA that was seen in the testis of untreated SCI rats and increased levels of ABP and SGP-2 mRNAs in SCI rats (P < 0.01). Follicle-stimulating hormone treatment of SCI rats did not affect FSHR mRNA levels by itself, but it blocked the stimulatory effect of T on FSHR and SGP-2 mRNAs. Fourteen days after SCI, testicular AR mRNA levels were not affected by T alone, but they increased in those rats that received FSH with or without concurrent T treatments (P < 0.05). In contrast to their effects in sham control rats, T or FSH alone or in combination resulted in significant increases in testicular levels of ABP, SGP-2, and FSHR mRNAs (P < 0.05). At this time, Trf mRNA in the testis of SCI rats was also suppressed by T (P < 0.05), as it did in sham control rats, but Trf mRNA was increased by the FSH (P < 0.01) that had inhibited this transcript in the testes of sham control rats. The effects of FSH on the Sertoli cell transcripts in SCI rats were either attenuated or blocked when T was given concurrently. In addition, testicular and serum T levels in those SCI rats that received FSH (alone or in combination with T) for 14 days were significantly increased, an effect that was not seen after sham surgery. These findings demonstrate that hormonal regulation of both Sertoli and Leydig cells was altered during the acute phase of SCI. Such changes may modify the functions of both cell types, thereby affecting the endocrine and/or paracrine microenvironment within the seminiferous epithelium. These effects could impair the functional capacity of Sertoli cells and contribute to impairment of spermatogenesis after SCI.

Androgen-induced calcium fluxes and gap junctional intercellular communication (GJIC) were studied in three different cell types. A transient (2–3 min duration) increase in intracellular calcium levels was observed within 20–30 sec of androgen addition, which was followed by a plateau phase with steroid concentrations higher than 1 nM. The kinetics of the calcium responses were similar in immature rat Sertoli cells, which contain normal nuclear receptors; the human prostatic tumor cell line, LNCaP, which contains a mutated nuclear receptor; and the human prostatic cell line, PC3, which does not contain a nuclear receptor. The human A431 tumor cell line did not respond to androgens. Concentrations of testosterone and the synthetic androgen, R1881, between 1–1000 pM induced transient calcium increases with ED₅₀ values near 1 pM and 1 nM, whereas dihydrotestosterone (DHT) was not active at these concentrations. At concentrations higher than 1 nM, testosterone, R1881, and DHT were equipotent in stimulating an increase in calcium that lasted for more than 10 min, with ED₅₀ values between 5 and 20 nM. Testosterone covalently bound to albumin was also active, whereas 11 related androstane compounds as well as progesterone and estradiol-17β were inactive at 1000 nM. The calcium response induced by the three androgens (10 nM) was abolished in all cell types by hydroxyflutamide (1000 nM) and finasteride (1000 nM), but not by cyproterone acetate (1000 nM). The calcium response was also abolished in the absence of extracellular calcium and strongly inhibited by the presence of verapamil. Exposure of the responsive cells to brief (150-sec) pulses of androgens generated calcium responses that were similar to those after continuous exposure. After exposure of Sertoli cells for only 30 sec to 100 nM testosterone, the calcium response lasted for at least 50 min. Although nuclear binding of androgens could be demonstrated, there was no evidence for tight binding to the plasma membrane under similar conditions. When protein synthesis was inhibited, an enhancement of GJIC between rat Sertoli cells, but not between LNCaP cells or PC3 cells, was observed within 15 min of the addition of 10 nM testosterone. Because nuclear androgens are not present in PC3 cells and many functional properties of the responsive system are different from the nuclear receptor in all three cell types, we postulate the existence of an alternative cell surface receptor system with biphasic response characteristics (high and low affinity). The calcium signals are probably coupled to the regulation of gap junctional efficiency between Sertoli cells. The low-affinity receptors may convey complementary androgen signals at elevated local levels such as in the testis, when nuclear receptors are (over)saturated.

A rise in intracellular calcium is the primary trigger for contractile activity in pregnant human myometrium. It is hypothesized that key proteins involved in myometrial calcium homeostasis are gestationally regulated and play an important role in the preparation for labor. The aims of the study were to investigate the role of sarcoplasmic reticulum Ca ATPases (SERCAs) in regulating spontaneous contractile activity in myometrium, and to determine the expression of SERCA isoforms 2a and 2b, and the plasma membrane Ca ATPase (PMCA), at term and during labor. Western blot analysis demonstrated that the expression of SERCA 2a and 2b significantly increased in myometrium of women in labor compared with those not in labor. The augmentation of contractile activity in laboring myometrium in the presence of a SERCA 2 inhibitor, cyclopiazonic acid (CPA), demonstrated the functional significance of this observation. It is interesting that the application of CPA in the presence of a calcium-activated potassium channel inhibitor to term nonlabor myometrium mimicked the response of myometrium from women in active labor to CPA alone. We conclude that the activity of SERCA isoforms becomes increasingly important in the maintenance of regular contractile activity during labor and may compensate for the functional loss of other calcium control pathways at term.

The B6.Y^TIR mouse fails to develop normal testes despite transcription of Sry, the primary testis-determining gene on the Y chromosome. Consequently, B6.Y^TIR fetuses with bilateral ovaries develop into apparently normal but infertile females. This infertility can be mainly attributed to oocyte incompatibility for postfertilization development. In addition, abnormality in preovulatory follicles and rapid loss of oocytes have been observed in XY ovaries. This study examined the effect of gonadotropins on follicular development and atresia in B6.Y^TIR prepubertal females. The results show that untreated XY females had fewer late preantral follicles and their frequency of atresia was lower. No other difference was found when they were compared with XX females. After treatment with gonadotropins for 24 h, frequency of atresia decreased in both XX and XY ovaries. After 48 h, most preovulatory follicles in XY ovaries were nonatretic, but the oocytes often were denuded. Immunocytochemical staining for connexin 43 detected punctate foci along the oocyte plasma membrane. The density of these foci changed during follicular development, which was similar in XX and XY ovaries. In conclusion, follicular development and atresia under the control of gonadotropins is not influenced by defective oocytes until the preovulatory phase.

The structure of the endozepine-like peptide (ELP) gene is closely related to the intracellular acyl-CoA binding protein (ACBP), but unlike the generalized distribution of the latter, it is restricted to the male germ cells of the testis. In the present study, a combination of nonradioactive in situ mRNA hybridization and immunohistochemistry was used to precisely determine the cellular expression patterns of ELP mRNA and protein in control and methoxyacetic acid (MAA)-treated rat testes. ELP transcripts are first detectable in late stages (step 6) of round spermatids, with transcription increasing through late-elongating steps. Translation of the ELP mRNA is delayed, with first immunohistochemical staining occurring in elongated spermatids at step 16, and protein accumulating through step 19. ELP immunoreactivity proves to be an excellent marker for late spermatid stages and highlights the presumably clonal recovery of spermatids following MAA treatment.

An endogenous circannual rhythm drives the seasonal reproductive cycle of a broad spectrum of species. This rhythm is synchronized to the seasons (i.e., entrained) by photoperiod, which acts by regulating the circadian pattern of melatonin secretion from the pineal gland. Prior work has revealed that melatonin patterns secreted in spring/summer entrain the circannual rhythm of reproductive neuroendocrine activity in sheep, whereas secretions in winter do not. The goal of this study was to determine if inability of the winter-melatonin pattern to entrain the rhythm is due to the specific melatonin pattern secreted in winter or to the stage of the circannual rhythm at that time of year. Either a summer- or a winter-melatonin pattern was infused for 70 days into pinealectomized ewes, centered around the summer solstice, when an effective stimulus readily entrains the rhythm. The ewes were ovariectomized and treated with constant-release estradiol implants, and circannual cycles of reproductive neuroendocrine activity were monitored by serum LH concentrations. Only the summer-melatonin pattern entrained the circannual reproductive rhythm. The inability of the winter pattern to do so indicates that the mere presence of a circadian melatonin pattern, in itself, is insufficient for entrainment. Rather, the characteristics of the melatonin pattern, in particular a pattern that mimics the photoperiodic signals of summer, determines entrainment of the circannual rhythm of reproductive neuroendocrine activity in the ewe.

Bradykinin and a number of peptide hormones such as angiotensin, endothelin, and vasopressin stimulate anion secretion in rat epididymis via local formation of PGE₂. These effects are mediated by cyclooxygenase (COX)-1 isozyme. The present study was undertaken to assess the androgen control of COX expression in the epididymis. Adult male Sprague-Dawley rats were bilaterally castrated through a scrotal route. Reverse transcription-polymerase chain reaction was used to measure COX-1 and COX-2 mRNAs in the epididymis in normal and castrated rats. Anion secretion in epithelia grown from the epididymides of these rats was studied by the short-circuit current technique. In normal rats, COX-1 and COX-2 mRNAs were detected in the intact epididymis. Elimination of spermatozoa by the technique of efferent duct ligation or flushing out spermatozoa did not affect the expression of either enzyme in the epididymis, indicating that the epithelium, but not spermatozoa, expressed the enzymes. Castration caused a time-dependent decrease in expression of COX-1 and COX-2 mRNAs, which were partially restored upon testosterone replacement. In epithelia cultured from castrated rats, there was a complete loss of bradykinin-induced anion secretion. This effect was reversible upon testosterone replacement. Although epithelia from castrated rats did not respond to bradykinin, they could respond to cAMP, forskolin, and PGE₂ with only 20% loss of response magnitude when compared with epithelia from normal rats. These results suggest that the expression of COX-1 and COX-2 are dependent on androgen. The loss of COX-1 expression after castration correlates with the specific loss of anion secretion induced by bradykinin and possibly other hormones.

Reversible phosphorylation is essential in regulating uterine contractions. Identification, characterization, and functional understanding of myometrium protein phosphatase(s) are lacking. Okadaic acid (OA), which inhibits protein phosphatase-1 (PP1) and PP2A, has been shown to alter uterine contractions. Experiments were conducted to determine the 1) identity of the myometrial OA-sensitive PP, 2) influence of OA on spontaneous and oxytocin (OT)-stimulated myometrial contractions, and 3) expression of uterine PPs during sexual development. Western blot analysis indicated the presence of PP1_α and PP2A in immature and mature mice. As determined by immunohistochemistry, gonadotropin-stimulated adult mouse uteri contain PP1_α in longitudinal and circular myometrial layers and endometrial epithelium. Conversely, PP2A was localized to the endometrial stroma. Cumulative addition of OA (n = 9; 10, 100, 250, 500, 1000 nM) did not significantly alter spontaneous contractions of mouse uterine horns in comparison to vehicle-treated controls (n = 9). By the end of the test period OA- and vehicle-treated uteri displayed a comparable decline in uterine contractions to 79.2% and 63.7%, respectively, of basal contractile activity. Pretreatment of uterine tissue with OA (1 μM; n = 7) significantly reduced contractile response to increasing concentrations of OT (8, 16, 32, 64 nM) in comparison to vehicle pretreatment (dimethyl sulfoxide; n = 7). At the end of the OT-administration period, contractile activity was 160.4% and 67.3% of basal contractile activity for vehicle (no OA) and OA-pretreated groups, respectively. During the early prepubertal period PP1_α was expressed in longitudinal myometrium and absent in circular myometrium; whereas, during the transition to sexual maturity PP1_α was observed in both the longitudinal and circular myometrium. In summary, these studies have indicated 1) that PP1 is the primary myometrial OA-sensitive PP; 2) that inhibition of PP1 had no effect on spontaneous contractions, whereas it markedly inhibited OT-stimulated uterine contractions; and 3) that PP1 is differentially expressed in the circular and longitudinal myometrium in relation to sexual development.

Greater than 95% of ovarian cancers originate in the epithelial cells on the surface of the ovary. The current study investigates the expression and action of transforming growth factor alpha (TGFα) in ovarian surface epithelium (OSE) and the underlying stroma in both normal and tumorigenic ovarian tissues. Normal bovine ovaries are used in the current study as a model system to investigate normal OSE functions. Transforming growth factor alpha and its receptor, the epidermal growth factor receptor (EGFR), were detected in the OSE from normal ovaries by immunocytochemistry (ICC). Ovarian stromal tissue also contained reduced but positive TGFα and EGFR immunostaining. To examine TGFα and EGFR gene expression, RNA was collected from normal bovine OSE and ovarian stromal cells. The TGFα and EGFR transcripts were detected in both fresh and cultured OSE and stromal cells by a sensitive quantitative reverse transcription polymerase chain reaction (QRT-PCR) assay. Transforming growth factor alpha gene expression was found to be high in freshly isolated OSE, but low in freshly isolated stroma. In contrast, EGFR expression was higher in the stroma compared to the OSE. Both the ICC and QRT-PCR indicate that normal OSE express high levels of TGFα in vivo and in vitro. In vitro, normal ovarian stromal cells develop the capacity to express high levels of EGFR. Human ovarian tumors from stage II, stage III, and stage IV ovarian cancer cases were found to express TGFα and EGFR protein in the epithelial cell component of the tumor by ICC analysis. The stromal cell component of human ovarian tumors contained little or no TGFα/EGFR immunostaining. Observations suggest that tumor progression may in part require autocrine stimulation of the epithelia. Transforming growth factor alpha was found to stimulate the growth of normal bovine OSE and stroma cells to the same level as epidermal growth factor. Two ovarian cancer cell lines, SKOV3 and OCC1, were also stimulated to proliferate in response to TGFα. Transforming growth factor alpha was also found to stimulate the expression of two growth factors previously shown to be produced by OSE. Transforming growth factor alpha stimulates both kit ligand/stem cell factor and keratinocyte growth factor production by OSE. The effect of hormones on TGFα and EGFR expression by the OSE was also examined. Human chorionic gonadotropin stimulated TGFα expression, but not FSH. Both hCG and FSH stimulated EGFR expression by OSE. Combined observations suggest a role of systemic hormones and a locally produced growth factor, TGFα, in OSE biology. Insight is also provided into how the OSE may develop abnormal growth characteristics involved in the onset and progression of ovarian cancer.

The calcium-dependent cell adhesion molecules, cadherins, regulate intercellular junction formation, cell sorting, and the establishment of cell polarity. Their important role in tissue remodeling suggests an involvement in ovarian cellular rearrangements throughout postnatal development. The ovary has a complex topology, and the ovarian follicle undergoes significant cellular rearrangements during its development. Cadherins have been detected previously in whole ovaries and in ovarian cells and cell lines with some immunolocalization in fetal and adult ovaries. This study examines the expression and localization of N- and E-cadherin throughout prepubertal ovarian and follicular development in the rat. We analyzed ovarian cadherin expression in rats from Day 19–20 of gestation to 25 days postpartum, during which follicle formation and folliculogenesis are the dominant ovarian events. Reverse transcriptase polymerase chain reaction detected N- and E-cadherin mRNA expression in the ovaries at all the ages examined. Semiquantification of Western blots of whole ovary extracts confirmed the presence of ovarian N- and E-cadherin protein at all ages with both showing peak expression at 7 days of age. Immunostaining revealed N- and E-cadherin expression in follicular and extrafollicular cell types, but only E-cadherin showed follicle-stage-dependent expression. The changes in cadherin expression, concurrent with ovarian growth and folliculogenesis, suggest a function for cadherins in the morphological and functional development of the prepubertal rat ovary.

The present study was conducted to examine the protective effect of cumulus cells on oocyte damage caused by reactive oxygen species (ROS), generated by the hypoxanthine-xanthine oxidase (XOD) system, during in vitro maturation of porcine oocytes. Cumulus-oocyte complexes (COCs) and cumulus-denuded oocytes (DOs) were cultured for 44 h in NCSU37 supplemented with cysteine, gonadotropins, 10% porcine follicular fluid, and hypoxanthine in the presence or absence of XOD. DNA cleavage and damage were analyzed using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and single cell microgel electrophoresis (comet) assay, respectively, and caspase-3 activity and glutathione (GSH) content were measured in each experimental group. Exposure of DOs to ROS resulted in meiotic arrest and the increase of degenerated oocytes. These degenerated DOs underwent apoptosis, as shown by the TUNEL-positive reaction within their germinal vesicles and the activation of caspase-3. The length of DNA migration in DOs treated with XOD was significantly longer than that of untreated DOs (P < 0.05). However, irreparable cell damage caused by ROS was not observed in COCs, and no difference was observed in the caspase-3 activity of both COCs treated with and without XOD. A significantly (P < 0.05) high level of GSH was found in COCs after 44 h of culture, compared with that of oocytes freshly isolated from their follicles, whereas GSH content in DOs markedly decreased after treatment with or without XOD. These findings suggest that cumulus cells have a critical role in protecting oocytes against oxidative stress-induced apoptosis through the enhancement of GSH content in oocytes.

A surgical procedure to aspirate follicular fluid concurrently from individual follicles from the same heifer was validated and used to determine if intrafollicular amounts of estradiol, progesterone, inhibins, activin-A, follistatins, and insulin-like growth factor binding proteins (IGFBP) differed for the future dominant compared with subordinate follicles during selection of the first wave dominant follicle. Heifers were subjected to surgery and aspiration of follicular fluid from the two or three largest follicles on Day 3 of the estrous cycle (∼1.5 days after emergence). Ultrasound was used to determine the fate of each aspirated follicle after surgery. At aspiration, diameter of the future dominant and largest subordinate follicle was similar in heifers. However, estradiol was higher, whereas IGFBP-4 was lower in the future dominant compared with the largest or next largest subordinate follicles. Also, the future dominant follicle in most cohorts had the highest estradiol and lowest IGFBP-4 compared with future subordinate follicles. We concluded that: IGFBP-4 and estradiol may have key roles in determining the physiological fate of follicles during selection of the first wave dominant follicle in heifers, and that both are reliable markers to predict which follicle in a growing cohort of 5- to 8.5-mm follicles becomes dominant.

Signals from the developing mammalian blastocyst rescue the corpus luteum (CL) and modulate the uterine environment in preparation for implantation and early pregnancy. Our previous studies demonstrated that both short- and long-term administration of chorionic gonadotropin (CG) markedly alters the morphology and the biochemical activity of the receptive endometrium. Because the effects of CG were superimposed on a progesterone-primed endometrium, this study was undertaken to determine if the inhibition of progesterone action by progesterone receptor antagonists (PRa) in intact and ovariectomized baboons would alter the action of CG on the endometrium at the time of uterine receptivity. In the short-term hCG-treated baboons, the PRa reduced the epithelial plaque reaction, completely inhibited α-smooth muscle actin (αSMA) expression in stromal fibroblasts, and induced the reappearance of the progesterone (PR) and estrogen (ERα) receptors in epithelial cells. However, this treatment protocol had no effect on the expression of glycodelin in the glandular epithelium. In contrast, glycodelin expression in addition to αSMA was suppressed in the ovariectomized animals. In the long-term hCG-treated baboons, the PRa had a similar effect on both αSMA, PR, and ER. In addition, this treatment also resulted in an inhibition of glycodelin expression in the glandular epithelium. These results indicate that blocking the action of progesterone on the endometrium even for a short period of time has a profound effect on the hCG-induced response in stromal fibroblasts. In contrast, for the diminution of glandular epithelial function in the presence of an ovary requires prolonged inhibition of progesterone action, suggesting a potential paracrine effect on the endometrium from the CL in response to hCG.

Little is known about the neuroendocrine control of fertility in the horse. In this species, unusual features characterize the normal estrous cycle such as a prolonged preovulatory LH surge during the follicular phase and a distinctive FSH surge during the midluteal phase. This study investigated the distribution and hormonal identity of gonadotrophs in the pars distalis (PD) and pars tuberalis (PT) of the equine pituitary gland as possible morphological bases for the referred unusual endocrine characteristics. In addition, the proportion of gonadotrophs in relation to other pituitary cell types during both the estrous cycle and anestrus were investigated. Pituitary glands were collected from sexually active (n = 5) and seasonally anestrous (n = 5) mares in November, and single or double immunofluorescent staining was carried out on 6-μm sections using monoclonal antibodies to the LHβ or FSHβ subunits and a polyclonal antibody to ovine LHβ. Gonadotrophs were densely distributed around the pars intermedia in the PD and in the caudal ventral region of the PT. In addition to isolated cells, clusters of gonadotrophs were found surrounding the capillaries. No significant differences were detected in the number of gonadotrophs between sexually active and anestrous mares in either the PD or PT. In the PD, gonadotrophs represented 22.7 ± 5.8% and 19.1 ± 2.1% of the total cell density in sexually active and anestrous animals, respectively (P > 0.05). However, in the PT, gonadotrophs accounted for a higher proportion of the total cell population in sexually active (6 ± 0.1%) than in anestrous (1.2 ± 0.05%) mares (P < 0.02). Double immunofluorescence revealed that the majority of gonadotrophs were bihormonal (i.e., positive for LH and FSH); however, in the sexually active mare, a larger proportion of gonadotrophs (22.5 ± 3.6%) were monohormonal for either LH or FSH, when compared to anestrous animals (9.7 ± 1.2%; P < 0.02). Based on these findings we conclude that: 1) although the relative distribution of gonadotrophs is similar to those reported for other species, a significantly larger proportion of gonadotroph cells is present in the equine pituitary gland; 2) gonadotroph density does not appear to differ between sexually active and anestrous mares in the PD; 3) a larger proportion of gonadotrophs is apparent in the PT of sexually active animals; and 4) although a large incidence of bihormonal gonadotrophs is present in the horse, specific LH or FSH cells differentiate predominantly during the sexually active phase.

The cystic fibrosis transmembrane conductance regulator (CFTR) or the small conductance cAMP-activated chloride channel encoded by the CFTR gene has been shown to play an important role in the formation of the epididymal fluid microenvironment. Mutation of the gene has led to widespread effects on male reproduction. Like other ion channels, CFTR is amenable to pharmacological intervention. Blocking CFTR in the epididymis could in principle lead to disruption of the epididymal fluid environment. We report for the first time two indazole compounds: lonidamine and 1-(2,4-dichlorobenzyl)-indazole-3-acrylic acid (AF2785) are potent blockers of CFTR in the epididymis. When added to the external solution under whole-cell patch clamp conditions, AF2785 and lonidamine inhibited the cAMP-activated chloride current in rat epididymal cells with apparent IC₅₀ values of 170.6 and 631.5 μM, respectively; by comparison the IC₅₀ value for diphenylamine-2-carboxylate, a well-known chloride channel blocker was 1294 μM. In cultured rat epididymal epithelia mounted in a Ussing chamber, AF2785 and lonidamine inhibited the cAMP-stimulated short-circuit current (a measure of chloride secretion) when added to the apical bathing solution with potency greater than any known chloride channel studied. It is proposed that in view of the important role CFTR plays in male reproduction, further study with these and other new indazole compounds for their CFTR blocking actions can provide a new avenue of research into the development of novel male contraceptives.

Cumulus cells and mural granulosa cells (MGC) are phenotypically different and there is now evidence suggesting that the oocyte plays an active role in determining the fate of follicular somatic cells. This study investigates the role of oocyte-secreted factor(s) in the regulation of the growth and differentiation of cumulus and MGC. Bovine cumulus-oocyte complexes (COC) and MGC were cultured with various hormones for 18 h followed by a further 6-h pulse of [³H]thymidine as an indicator of follicular cell DNA synthesis. The COC incorporated 11 to 14 times more [³H]thymidine than MGC in either the absence or presence of 50 ng/ml insulin-like growth factor (IGF)-I. Purified porcine FSH (450 ng/ml) added together with IGF-I marginally increased ³H incorporation in MGC relative to IGF-I alone but dramatically decreased incorporation in COC sixfold. Conversely, mean progesterone production in the presence of IGF-I FSH was 13-fold higher from MGC than from COC, confirming a distinctive phenotype of cumulus cells. However, this phenotype was found to be dependent on the presence of the oocyte, as microsurgical removal of the oocyte (oocytectomy) resulted in an 11-fold decrease in [³H]thymidine incorporation in cumulus cells treated with IGF-I, elimination of the inhibitory effect of FSH on IGF-I-stimulated DNA synthesis, and led to a 2-fold increase in progesterone production in medium with IGF-I and FSH. All of these markers were completely restored to COC levels when oocytectomized complexes were cocultured with denuded oocytes (DO) at a concentration of 0.5 oocytes/μl, demonstrating that oocytes secrete a soluble factor(s) that promotes growth and attenuates cumulus cell progesterone secretion. In the presence of IGF-I, [³H]thymidine incorporation in MGC increased ninefold above control levels with the addition of DO. The addition of FSH to IGF-I-increased ³H counts in MGC, however, led to a decrease in counts in MGC DO as is also observed in COC. Furthermore, progesterone production was halved when DO were added to MGC cultures, most notably in the presence of IGF-I and/or FSH. These results provide further evidence that MGC and cumulus cells have distinctive phenotypes and that the oocyte is responsible for some of the characteristic features of cumulus cells. Bovine oocytes secrete a soluble factor(s) that simultaneously promotes growth and attenuates steroidogenesis in follicular somatic cells.

Intra-amniotic infection leads to preterm labor and is associated with the local release of inflammatory cytokines by fetal membranes, resulting in the production of uterotonic prostaglandins. Oxytocin, however, also plays a key role in the initiation of labor. Short-term exposure of myometrium to interleukin (IL)-1 enhances oxytocin signaling and contractility. With intrauterine infection, however, myometrium is exposed to inflammatory cytokines for prolonged periods. The present study was conducted to demonstrate that myometrial oxytocin signaling is significantly impaired following prolonged exposure to IL-1. Myometrial cells were treated with IL-1 for 24 h. Oxytocin-stimulated inositol trisphosphate (IP₃) production was measured in tritiated myoinositol-loaded myometrial cells. Arachidonic acid (AA) release was measured in tritiated AA-loaded myometrial cells. Increases in intracellular calcium were measure with fluo-3. Prostaglandin (PG) F_2α and 6-keto-PGF_1α were measured by ELISA assay. Prolonged exposure of myometrial cells to IL-1 resulted in a significant reduction in oxytocin-mediated signaling as measured by IP₃ production and AA release, as well as a decrease in intracellular calcium. Prolonged exposure of myometrial cells to IL-1, however, resulted in enhanced PG release. Oxytocin may not contribute significantly to the labor-inducing action of IL-1 in the setting of preterm labor with prolonged infection.

Programmed cell death (apoptosis) occurs in nearly all cell types examined, including mammalian oocytes and embryos, where it may underlie some forms of infertility in humans. Although the molecular machinery participating in apoptosis have been intensely investigated, the accompanying physiological changes have not received similar attention. In this study, a novel electrophysiology technique has been employed to monitor real-time perturbations in the physiology of mouse embryos undergoing apoptosis evoked by hydrogen peroxide, diamide, and staurosporine. Despite differences in their mode of action, these agents evoked a similar early change in cellular physiology; namely, a pronounced, transient, potassium efflux through tetraethylammonium-sensitive potassium channels accompanied by cell shrinkage. Mouse zygotes exposed to 200 μM H₂O₂ exhibited potassium efflux that elevated the potassium concentration of the media surrounding embryos by 1.4 ± 0.1 μM. Pretreatment with tetraethylammonium inhibited this increase (0.2 ± 0.1 μM). Our results indicate that potassium efflux through potassium channels and concurrent cell shrinkage are early indicators of cell death in embryos and that noninvasive measurements of potassium pathophysiology may identify embryos undergoing cell death prior to the manifestation of other morphological or molecular hallmarks of cell death.

Angiogenesis is the process that drives blood vessel development in growing tissues in response to the local production of angiogenic factors. With the present research the authors have studied vascular endothelial growth factor (VEGF) production in ovarian follicles as a potential mechanism of ovarian activity regulation. Prepubertal gilts were treated with 1250 IU equine chorionic gonadotropin (eCG) followed 60 h later by 750 IU of human chorionic gonadotropin (hCG) in order to induce follicle growth and ovulation. Ovaries were collected at different times of the treatment and single follicles were isolated and classified according to their diameter as small (<4 mm), medium (4–5 mm), or large (>5 mm). VEGF levels were measured in follicular fluid by enzyme immunoassay, and VEGF mRNA content was evaluated in isolated theca and granulosa compartments. Equine chorionic gonadotropin stimulated a prompt follicular growth and induced a parallel evident rise in VEGF levels in follicular fluid of medium and large follicles. Analysis of VEGF mRNA levels confirmed the stimulatory effect of eCG, showing that it is confined to granulosa cells, whereas theca cells maintained their VEGF steady state mRNA. Administration of hCG 60 h after eCG caused a dramatic drop in follicular fluid VEGF that reached undetectable levels in 36 h. A parallel reduction in VEGF mRNA expression was recorded in granulosa cells. The stimulating effect of eCG was also confirmed by in vitro experiments, provided that follicles in toto were used, whereas isolated follicle cells did not respond to this hormonal stimulation. Consistent with the observation in vivo, granulosa cells in culture reacted to hCG with a clear block of VEGF production. These results demonstrate that while follicles of untreated animals produce stable and low levels of the angiogenic factor, VEGF markedly rose in medium and large follicles after eCG administration. The increasing levels, essentially attributable to granulosa cells, are likely to be involved in blood vessel development in the wall of growing follicles, and may play a local key role in gonadotropin-induced follicle development. When ovulation approaches, under the effect of hCG, the production of VEGF is switched off, probably creating the safest conditions for the rupture of the follicle wall while theca cells maintained unaltered angiogenic activity, which is probably required for corpus luteum development.

The effects of insulin-like growth factor I (IGF-I) and insulin on the function of coho salmon gonadotropes in vitro were investigated. Dispersed pituitary cells from immature coho salmon (Oncorhynchus kisutch) were incubated with IGF-I for 1, 3, 7, or 10 days, then incubated with salmon GnRH for an additional 24 h. Medium FSH content before and after GnRH treatment and intracellular FSH content after GnRH treatment were measured. Incubation of pituitary cells with IGF-I for 7 or 10 days increased GnRH-stimulated FSH release and remaining cell content, but did not affect basal release. To examine the specificity of the effects of IGF-I, we compared FSH release and cell content of FSH and LH after 10-day incubation with a range of concentrations of IGF-I or insulin. Incubation with physiological concentrations of IGF-I resulted in significantly higher GnRH-stimulated FSH release and remaining cell content of FSH and LH. Conversely, supraphysiological concentrations of insulin were required to produce more moderate effects on gonadotropin levels. These results suggest that elevation of gonadotropin levels by IGF-I may be one mechanism by which somatic growth and nutrition promote pubertal development in salmon.

Testosterone at physiological levels cannot exert negative feedback action on LH secretion in long-term castrated male monkeys. The cellular basis of this refractoriness is unknown. To study it, we compared two groups of male rhesus macaques: one group (group 1, n = 4) was castrated and immediately treated with testosterone for 30 days; the second group (group 2, n = 4) was castrated and treated with testosterone for 9 days beginning 21 days after castration. Feedback control of LH by testosterone in group 1 was normal, whereas insensitivity to its action was found in group 2. Using the endpoints of concentrations of aromatase activity (P450_AROM messenger RNA [mRNA]) and androgen receptor mRNA in the medial preoptic anterior hypothalamus and in the medial basal hypothalamus, we found that aromatase activity in both of these tissues was significantly lower, P < 0.01, in group 2 compared with group 1 males. P450_AROM mRNA and androgen receptor mRNA did not differ, however. Our data suggest that the cellular basis of testosterone insensitivity after long-term castration may reside in the reduced capacity of specific brain areas to aromatize testosterone. Because P450_AROM mRNA did not change in group 2 males, we hypothesize that an estrogen-dependent neural deficit, not involving the regulation of the P450_AROM mRNA, occurs in long-term castrated monkeys.

In order to better understand how tumor necrosis factor (TNF)-α may contribute to the local regulation of uterine cell death, cultures of mouse uterine epithelial WEG-1 cells were exposed to TNF-α and observed at different time intervals. Earliest decrease in cell viability was observed after 31 h of exposure to 50 ng/ml mouse TNF-α and was associated with the expression of several markers of apoptosis. Treatment with human TNF-α or addition of a neutralizing antibody against TNF-α receptor protein 80 to mouse TNF-α resulted in attenuated induction of apoptosis, suggesting that coengagement of the two TNF-α receptor types is required for maximal impact. Ceramide analogs failed to replicate the effect of TNF-α and the stress-activated protein kinase signaling pathway was not activated by the cytokine. Treatment with mouse TNF-α resulted in an increase in nuclear factor (NF)κB activity that receded after 24 h. The impact of human TNF-α on NFκB activation was more moderate. Addition of either one of three different inhibitors of NFκB (SN50, PDTC, and A771726) to mouse TNF-α sensitized WEG-1 cells to the toxicity of the cytokine. Our data suggest that WEG-1 cells initiate their response to TNF-α with an increase in NFκB activation that may have transiently biased these cells toward cell death resistance.

One of the problems associated with in vitro culture of primordial germ cells (PGCs) is the large loss of cells during the initial period of culture. This study characterized the initial loss and determined the effectiveness of two classes of apoptosis inhibitors, protease inhibitors, and antioxidants on the ability of porcine PGCs to survive in culture. Results from electron microscopic analysis and in situ DNA fragmentation assay indicated that porcine PGCs rapidly undergo apoptosis when placed in culture. Additionally, α₂-macroglobulin, a protease inhibitor and cytokine carrier, and N-acetylcysteine, an antioxidant, increased the survival of PGCs in vitro. While other protease inhibitors tested did not affect survival of PGCs, all antioxidants tested improved survival of PGCs (P < 0.05). Further results indicated that the beneficial effect of the antioxidants was critical only during the initial period of culture. Finally, it was determined that in short-term culture, in the absence of feeder layers, antioxidants could partially replace the effect(s) of growth factors and reduce apoptosis. Collectively, these results indicate that the addition of α₂-macroglobulin and antioxidants can increase the number of PGCs in vitro by suppressing apoptosis.

Leydig cells in the adult rat testis differentiate during the neonatal-prepubertal period. However, the stimulus for the initiation of their differentiation is still not clear. In the present study our objectives were to test the effects of thyroid hormone and LH on the initiation of precursor cell differentiation into Leydig cells in the prepubertal rat testis. Four groups of Sprague-Dawley rats were used. All treatments began at postnatal Day 1. Rats in groups I, II, and III received daily s.c. injections of saline (200 μl, controls), triiodothyronine (T₃, 50 μg/kg body weight, hyperthyroid), and LH (ovine LH 10 μg/rat/day), respectively. Rats in group IV were made hypothyroid from postnatal Day 1 by adding 0.1% propylthiouracil (PTU) to their mother's drinking water. Testes of rats were collected at 7, 8, 9, 10, 11, 12, 16, and 21 days of age, fixed in Bouin's solution, and embedded in paraffin for immunocytochemical studies. Immunoexpression of 3β-hydroxysteroid dehydrogenase (3β-HSD) and LH receptors (LHR) in testicular interstitial cells (other than the fetal Leydig cells) was observed using the avidin-biotin method. In control rats, out of all spindle-shaped cell types in the testis interstitium, only the peritubular mesenchymal cells showed positive immunolabeling for 3β-HSD, beginning from the postnatal Day 11. However, positive immunolabeling for LHR was first detected in these cells at Day 12, i.e., after acquiring the steroidogenic enzyme activity. In T₃-treated rats 3β-HSD positive spindle-shaped cells were first observed at Day 9 (i.e., 2 days earlier than controls), and LHR-positive cells were first observed on Day 11 (2 days later than obtaining 3β-HSD immunoactivity); they were exclusively the peritubular mesenchymal cells. The 3β-HSD- and LHR-positive spindle-shaped cells were absent in the testis interstitium of LH-injected rats from Days 7 through 12 but were present at postnatal Day 16. In addition, more fetal Leydig cell clusters and fetal Leydig cells in mitosis were present in LH-treated rats compared to rats in all other treatment groups. Following their first detection, the number of positive cells for each protein continued to increase at each subsequent age in controls, T₃-, and LH-injected groups. In PTU rats, 3β-HSD and LHR-positive spindle-shaped cells were absent throughout the experimental period. From these observations, it is possible to suggest the following regarding the developing rat testis interstitium. 1) The precursor cells for the adult generation of Leydig cells in the postnatal rat testis are the peritubular mesenchymal cells. 2) Luteinizing hormone does not initiate the onset of mesenchymal cell differentiation into Leydig cells, instead it delays this process. However, daily LH treatment causes mitosis in fetal Leydig cells and increase in fetal Leydig cell clusters. 3) Thyroid hormone is critical to initiate the onset of mesenchymal cell differentiation into adult Leydig cells.

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is a multifunctional protein expressed in the uterus of essentially all species, yet the function of this protein is uncertain. To assess the role of TIMP-1 in the uterine events that occur during the murine estrous cycle, mature female TIMP-1 wild-type and null mice were monitored for reproductive cyclicity. Mice were sacrificed in each stage of the estrous cycle, and peripheral blood was collected and assayed for serum estradiol and progesterone content by RIA. Uterine morphology and TIMP-1, TIMP-2, TIMP-3, and TIMP-4 mRNA expression were also examined between genotypes in each stage of the estrous cycle. Disruption of the TIMP-1 gene product was associated with an altered reproductive cycle characterized by a significant decrease in the length of the estrus period in the null mice. Also during the period of estrus, null mice expressed significantly lower levels of uterine TIMP-3 mRNA expression, altered uterine morphology, significantly higher serum estradiol levels, and significantly lower serum progesterone levels compared to their wild-type counterparts. It is concluded from this study that TIMP-1 has a multifaceted role in regulating the murine reproductive cycle, and this control appears to be at the level of both the uterus and the ovary.

Proteoglycans (PGs) consist of a core protein and attached glycosaminoglycans (GAGs) and have diverse roles in cell and tissue biology. In follicles PGs have been detected only in follicular fluid and in cultured granulosa cells, and the composition of their GAGs has been determined. To identify PGs in whole ovarian follicles, not just in follicular fluid and granulosa cells, small (1–3-mm) bovine follicles were harvested. A proportion of these was incubated with ³⁵SO₄ for 24 h to incorporate radiolabel into the GAGs. The freshly harvested and cultured follicles were sequentially extracted with 6 M urea buffer, the same buffer with 0.1% Triton X-100 and then with 0.1 M NaOH. Proteoglycans were subjected to ion-exchange and size-exclusion chromatography. The GAGs were analyzed by chemical and enzymic digestion, and on the basis of their composition, we chose a list of known PGs to measure by ELISA analyses. Versican, perlecan, decorin, but not aggrecan or biglycan, were identified. These, excluding decorin for technical reasons, as well as a basal lamina glycoprotein, nidogen/entactin, were immunolocalized. Versican was localized to the thecal layers, including externa and the interna particularly in an area adjacent to the follicular basal lamina. Perlecan and nidogen were localized to the follicular basal lamina of antral follicles, both healthy and atretic, but not to that of preantral follicles. Both were localized to subendothelial basal laminas, but the former was not readily detected in arteriole smooth muscle layers. This study has confirmed the presence of versican and perlecan, but not the latter as a component of follicular fluid, and identified decorin and nidogen in ovarian antral follicles.

We previously described a putative creatine kinase M isoform in human sperm that is developmentally regulated and expressed during late spermiogenesis, simultaneous with cytoplasmic extrusion. We have now identified this protein as the testis-expressed 70-kDa heat shock protein chaperone known as HspA2 (the human homologue of mouse Hsp70-2). We have isolated and characterized HspA2 (formerly CK-M) by amino acid sequencing and have localized it by immunocytochemistry to spermatocytes at low levels, to spermatids, and in the tail of mature sperm. The specificity of the CK-M/HspA2 antiserum to HspA2 was demonstrated on immunoblots of one- and two-dimensional SDS-PAGE. In agreement with our earlier biochemical data, immunocytochemistry of testicular tissue indicated that HspA2 is selectively expressed in mature spermatids and in sperm about to be released in the seminiferous tubuli. The identity of HspA2 has been further confirmed by cross-absorption of the mouse HSP70-2 antibody by the HspA2/CK-M fraction, and by identical immunostaining patterns of human testicular tissue using either the anti-CK-M/HspA2 or an anti-mouse Hsp70-2 antisera. During spermiogenesis, both cytoplasmic extrusion and plasma membrane remodeling, which facilitate the formation of the zona pellucida binding site, involve major intrasperm protein transport, which may be chaperoned by HspA2. Accordingly, in immature human sperm, which fail to express HspA2, there is cytoplasmic retention and lack of zona pellucida binding. The present findings provide the biological rationale for the role of the human HspA2 as an objective biochemical marker of sperm function and male fertility, which we have established in earlier clinical studies.

The regulation of expression of cyclooxygenase 2 (COX-2) was investigated by treatment with PGE₂ in human endometrial adenocarcinoma cell line HEC-1B. One μM PGE₂ could stimulate the expression of COX-2 approximately twofold in this cell line. The same concentration of PGE₂ also stimulated activation of mitogen-activated protein kinase (MAP kinase) and protein kinase B (PKB). PGE₂-induced MAP kinase activation was sensitive to a MAP kinase kinase (MEK) inhibitor, PD098059, and a protein kinase A inhibitor, H-89. PD098059 and H-89 also partially inhibited the expression of COX-2 stimulated by PGE₂. PGE₂ could stimulate the activation of PKB, which was sensitive to phosphatidylinositol-3-OH kinase (PI3K) inhibitor, wortmannin. Whereas wortmannin alone partially inhibited the expression of COX-2, a combination of wortmannin and PD098059 totally inhibited PGE₂-mediated COX-2 expression. These results suggest that MAP kinase and PI3K pathways are stimulated with PGE₂, and that both of these pathways are involved in the expression of COX-2. In addition, they also suggest that protein kinase A remains upstream of PGE₂-induced activation of MAP kinase in HEC-1B cells.

Ovarian follicular growth and steroidogenesis are controlled by the interaction of insulin-like growth factors (IGFs) and gonadotropins. The objective was to determine the temporal and spatial relationships for gonadotropin receptor, steroidogenic enzyme, and IGF system gene expression during the development of preovulatory porcine follicles. Sows (n = 18) were weaned and follicles were monitored by transrectal ultrasonography. Ovaries were collected from sows when the mean diameter of the preovulatory follicular cohort was approximately 2, 4, 6, or 8 mm. mRNA were measured by in situ hybridization for individual follicles within the preovulatory cohort (3 to 5 follicles per sow). Patterns of gene expression detected by in situ hybridization were confirmed by RNase protection analyses of pooled RNA samples. The amount of LH receptor mRNA and steroidogenic enzyme mRNA (17α-hydroxylase and aromatase) increased as the mean diameter of the follicular cohort increased from 2 to 6 mm, but then decreased abruptly for 8-mm follicles. Estradiol concentrations in follicular fluid closely followed the expression patterns of steroidogenic enzymes and LH receptor mRNA. FSH receptor mRNA was present in cohorts of 2-mm follicles but declined in 4-mm follicles and was undetectable in 6- and 8-mm follicles. The expression of IGF-I and type I IGF receptor mRNA were similar for follicles of 2, 4, 6, and 8 mm. In contrast, IGF-II mRNA progressively increased in follicles collected from 2-, 4-, and 6-mm cohorts, and then decreased slightly at 8 mm. Type II IGF receptor mRNA was greatest in 8-mm follicles. IGF binding protein-2 (BP-2) mRNA decreased as follicles achieved progressively larger sizes during the preovulatory period (2 to 8 mm), whereas the IGFBP-4 mRNA remained relatively low for follicles in 2- to 6-mm cohorts but then increased markedly in 8-mm follicles. In summary, temporal and spatial patterns of gene expression for gonadotropin receptor, steroidogenic enzyme, and IGF system genes were characterized in preovulatory porcine follicles by using in situ hybridization and RNase protection analyses. The unique patterns of gene expression suggest interdependence among specific genes that may be essential for preovulatory follicular development.

Twenty-five pregnant Dorsett ewes were randomly divided into three groups to test if ewes use their vomeronasal organs for offspring recognition during nursing. One group of eight ewes (procaine) were made anosmic by irrigation of the nasal olfactory apparatus with a zinc sulphate procaine solution. The second group of nine ewes (cauterized) had their vomeronasal organs rendered nonfunctional by cauterization of the nasoincisive duct. The third group of eight ewes were the controls. Parturition was synchronized in all ewes with betamethasone on Day 145 of gestation. Maternal responsiveness was tested two separate times with 1- to 2-day-old alien lambs. Each alien lamb trial was conducted 24 h apart. Cauterized ewes allowed alien lambs to suckle and they were unable to distinguish alien lambs from their own lambs, whereas the ewes in both groups with functional vomeronasal organs (procaine and control) violently rejected any alien lamb's attempt to suckle. Thus, female sheep use their vomeronasal organs for neonatal offspring recognition.

Fibroblast growth factor-10 (FGF-10) is a stromal-derived paracrine growth factor considered to be important during embryogenesis; however, its expression by cells in the female reproductive tract has not been investigated. Therefore, an ovine FGF-10 cDNA was cloned from an ovine endometrial cDNA library to investigate expression and potential paracrine characteristics of FGF-10 in the ovine uterus. The ovine FGF-10 cDNA encodes a protein of 213 amino acids and possesses an unusually long 5′ untranslated region (UTR). In situ hybridization demonstrated that ovine FGF-10 mRNA was expressed by endometrial stromal cells and by mesenchymal cells of the chorioallantoic placenta. The mRNA for FGF-7, a homologue of FGF-10, was localized in the tunica muscularis of blood vessels in endometrium and myometrium. In contrast, FGF receptor 2IIIb, the high-affinity receptor for both FGF-10 and FGF-7, was expressed exclusively in luminal epithelium, glandular epithelium, and placental trophectoderm. The in vivo spatial expression pattern suggests that FGF-10 is a novel endometrial stromal cell-derived mediator of uterine epithelial and conceptus trophectodermal functions. The nonoverlapping spatial patterns of expression for FGF-10 and FGF-7 in ovine uterus and conceptus suggest independent roles in uterine function and conceptus development.