Analysis of spatial and temporal expression patterns of bone morphogenetic protein family members in the rat uterus over the estrous cycle.

Recent studies have demonstrated that bone morphogenetic proteins (BMPs) play fundamental roles in female fertility. This is particularly evident in terms of the ovary. One major question that is just beginning to be addressed is the role of BMPs in the non-pregnant uterus. To help fill this gap, we used in situ hybridization to investigate the expression of BMP family members in the rat uterus over the estrous cycle. We found that the endometrial/uterine cycle is accompanied by the expression of several components of the BMP pathway - including ligands, receptors and antagonists. The mRNAs encoding BMP receptors are expressed in the epithelial (BMP-RIA, -RIB and -RII), periluminal stroma (BMP-RIA and -RII) and smooth muscle cells (BMP-RIA and -RII). The expression of all three receptors showed clear cyclic variations. The mRNAs encoding BMP ligands were highly expressed in the periluminal stroma (BMP-2 and -7) and glandular epithelium (BMP-7). The expression of BMP-2, but not BMP-7, was cyclical. Notably, the periluminal stroma expressed noggin mRNA. In the blood vascular system, BMP-4, -6 and -RII mRNAs were expressed in myometrial endothelial cells. Interestingly, follistatin, noggin, and BMP-4, -6 and -7 mRNAs were expressed in eosinophilic leukocytes, suggesting unexpected roles for eosinophil-derived BMPs in uterine function. We conclude that BMP ligands, receptors and antagonists are expressed in spatially and temporally restricted patterns that are consistent with a physiological role for these regulatory molecules in promoting uterine cellular processes including cell proliferation, differentiation and apoptosis during the cycle.

The role of bone morphogenetic proteins in ovarian function.

Bone morphogenetic proteins (BMPs) represent the largest subclass of growth factors in the transforming growth factor beta (TGF-beta) superfamily. BMPs have proven to be multifunctional regulators of a wide variety of biological processes in numerous types of cell and tissue. The role of inhibins, activins and TGF-betas (which also belong to the TGF-beta superfamily) in reproduction has been studied extensively over the last 15 years. However, there were no reports on the role of BMPs in the mammalian ovary until 1999 when we reported an intrinsic ovarian BMP system replete with BMP ligands, receptors and novel biological functions. Since this report it has become clear that the BMP system plays an important role in the regulation of ovarian function, evidenced by the ability of BMPs to control granulosa cell proliferation and cytodifferentiation, as well as oocyte development. The physiological relevance of the BMP system has recently been highlighted by the discovery that genetic mutations in the BMP-15 ligand or the BMP type IB receptor lead to critical aberrations in folliculogenesis and ovulation. This review provides a current overview of the rapidly emerging field of the BMP system in mammalian ovarian function.

The physiology of folliculogenesis: the role of novel growth factors.

OBJECTIVE: To assess major physiological events underlying folliculogenesis, including FSH-dependent dominant follicle (DF) formation, LH/hCG signaling, and the role of novel regulatory molecules in these developmental processes. DESIGN: Review of some of the past and recent advances in ovarian biology, focusing attention on [1] two novel oocyte-derived growth factors, growth differentiation factor-9 (GDF-9) and bone morphogenetic protein (BMP-15); and [2] a recently discovered follicular insulin-like growth factor binding protein-4 (IGFBP-4) protease, pregnancy-associated plasma protein-A (PAPP-A), that can degrade the FSH antagonist IGFBP-4. RESULT(S): Oocyte-derived GDF-9 and BMP-15 are obligatory for folliculogenesis and female fertility in laboratory animals through their ability to stimulate granulosa cell proliferation and modulate FSH-dependent cytodifferentiation. The expression of these growth factors in human primary oocytes supports the hypothesis that GDF-9 and BMP-15 could be involved in ovary function in women. Pregnancy-associated plasma protein-A is a marker for the human dominant follicle and its product the corpus luteum, raising the possibility that this putative FSH antagonist might regulate FSH bioactivity during folliculogenesis and luteogenesis. CONCLUSION(S): Oocyte-derived and granulosa-derived regulatory proteins perform very important functions in FSH-dependent folliculogenesis. The current challenges are to understand the role of these novel proteins in ovary physiology and pathophysiology in women.

Role of growth factors in ovary organogenesis.

The organogenesis of the ovary encompasses the formation of a great variety of structures, both germinal and nongerminal. Primordial follicle (PF) formation is of the utmost importance because PFs are obligatory for the reproductive cycle and female fertility. The major events involved in PF formation are described. Areas that could benefit from more investigation are discussed. The working premise is that the number of PFs formed during normal ovary organogenesis varies from one female to the next (ranging from high to low), and that this variability is revealed by the timing of age-related infertility and the menopause. Implicit in this supposition is the concept that anything that alters the sequence of events involved in the process of PF development will have important consequences on female fertility and health.

Bone morphogenetic protein-15 inhibits follicle-stimulating hormone (FSH) action by suppressing FSH receptor expression.

We have recently reported that oocyte-derived bone morphogenetic protein-15 (BMP-15) can directly modulate follicle-stimulating hormone (FSH) action in rat granulosa cells. Here, we investigate underlying mechanisms of this BMP-15 effect. Treatment with BMP-15 alone exerted no significant effect on the basal expression of mRNAs encoding steroidogenic acute regulatory protein, P450 side chain cleavage enzyme, P450 aromatase, 3beta-hydroxysteroid dehydrogenase, luteinization hormone receptor, and inhibin/activin subunits. However, BMP-15 markedly inhibited the FSH-induced increases in these messages. In striking contrast, BMP-15 did not change the forskolin-induced levels of these transcripts. Thus, the inhibitory effect of BMP-15 on FSH action must be upstream of cAMP signaling. We next examined changes in FSH receptor mRNA expression. Interestingly, BMP-15 severely reduced the levels of FSH receptor mRNA in both basal and FSH-stimulated cells. To determine whether this effect was at the level of FSH function, we investigated the effect of BMP-15 on FSH bioactivity. Consistent with the mRNA data, BMP-15 inhibited the biological response of FSH, but not that of forskolin. Based on these results, we propose that BMP-15 is an important determinant of FSH action through its ability to inhibit FSH receptor expression. Because FSH plays an essential role in follicle growth and development, our findings could have new implications for understanding how oocyte growth factors contribute to folliculogenesis.

Bone morphogenetic protein-15. Identification of target cells and biological functions.

In developing ovarian follicles, the regulation of cell proliferation and differentiation is tightly coordinated. Precisely how this coordination is achieved is unknown, but recent observations have suggested that molecules emitted by the oocyte are involved in the process. The newly discovered oocyte-specific growth factor, bone morphogenetic protein-15 (BMP-15), is one such molecule. At present, nothing is known about the target cells and biological functions of BMP-15. To fill this gap in our knowledge, recombinant BMP-15 and its antibody were produced and used to determine BMP-15 expression and bioactivity. BMP-15 mRNA and protein were shown to be co-expressed in oocytes throughout folliculogenesis, supporting the idea that BMP-15 is a physiological regulator of follicle cell proliferation and/or differentiation. To test this, we used primary cultures of rat granulosa cells (GCs). We found that BMP-15 is a potent stimulator of GC proliferation, and importantly, the mitogenic effect was follicle-stimulating hormone (FSH)-independent. By contrast, BMP-15 alone had no effect on steroidogenesis. However, it produced a marked decrease in FSH-induced progesterone production, but had no effect on FSH-stimulated estradiol production. This result indicates that BMP-15 is a selective modulator of FSH action. In summary, this study identifies GCs as the first target cells for BMP-15. Moreover, it identifies the stimulation of GC proliferation and the differential regulation of two crucial steroid hormones as the first biological functions of BMP-15. Significantly, BMP-15 is the first growth factor that can coordinate GC proliferation and differentiation in a way that reflects normal physiology.

The role of the oocyte in folliculogenesis.

Novel regulatory proteins have been identified within oocytes that are crucially involved in folliculogenesis. One of the most exciting oocyte signaling molecules is a novel member of the transforming growth factor beta (TGF-beta) superfamily, growth differentiation factor 9 (GDF-9). Loss-of-function studies have established that GDF-9 is obligatory for proper folliculogenesis and fertility in female mice. The current challenges are to understand how oocyte morphogens regulate folliculogenesis and how their actions and interactions are integrated into the overall processes of physiology and pathophysiology. Who would have thought that oocyte morphogens would be so crucial for reproduction?

Changes in insulin-like growth factor-binding protein-3 messenger ribonucleic acid in endothelial cells of the human corpus luteum: a possible role in luteal development and rescue.

In the human menstrual cycle, extensive angiogenesis accompanies luteinization; and the process is physiologically important for corpus luteum (CL) function. During luteolysis, the vasculature collapses, and the endothelial cells die. In a conceptual cycle, the CL persists both functionally and structurally beyond the luteoplacental shift. Although luteal rescue is not associated with increased angiogenesis, endothelial survival is extended. Despite the central role of the luteal vasculature in fertility, the mechanisms regulating its development and demise are poorly understood. There is increasing evidence that insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) may be important effectors of luteal function. Here, we have found that IGFBP-3 messenger RNA is expressed in the endothelium of the human CL and that the levels of message change during luteal development and rescue by human CG. The signal was strong during the early luteal phase, but it showed significant reduction during the mid- and late luteal phases. Interestingly, administration of human CG caused a marked increase in the levels of IGFBP-3 messenger RNA in luteal endothelial cells that was comparable to that observed during the early luteal phase. We conclude that endothelial cell IGFBP-3 expression is a physiological property of the CL of menstruation and pregnancy. These observations raise the intriguing possibility that the regulated expression of endothelial IGFBP-3 may play a role in controlling angiogenesis and cell responses in the human CL by autocrine/paracrine mechanisms.

Pregnancy-associated plasma protein-A gene expression in human ovaries is restricted to healthy follicles and corpora lutea.

Recently, Pregnancy-Associated Plasma Protein-A (PAPP-A) in human follicular fluid was identified as an insulin-like growth factor binding protein-4 protease (IGFBP-4ase). The ability of IGFBP-4ase to inactivate the FSH antagonist, IGFBP-4, has suggested a possible role for PAPP-A in regulating FSH action. Despite growing interest in this protease, the question of whether the PAPP-A gene is expressed in ovaries of normal cycling women is unknown. To fill this basic gap in our knowledge, we have identified the cellular sites of PAPP-A gene expression in normal human ovaries by in situ hybridization. PAPP-A mRNA was low or undetectable in preantral follicles, small (1-2 mm) healthy and atretic antral follicles, larger atretic antral follicles, surface epithelium, tunica albuginea and connective tissue cells. In contrast, an intense PAPP-A hybridization signal was evident in the healthy antral follicles examined from 5 mm to the preovulatory stage. In these follicles, the signal was restricted to the granulosa cells (GC). An intense signal for PAPP-A mRNA was also present in healthy corpora lutea (CL), being localized to a subset of large luteal cells. Collectively, these results provide the first evidence that the gene encoding PAPP-A is expressed in ovaries of normal cycling women and show that the gene is expressed almost exclusively in healthy GC and CL cells. The restricted pattern of PAPP-A expression in normal human ovaries suggests that PAPP-A may be a functional marker of the dominant follicle and its product, the CL. Although the physiological function of ovarian PAPP-A remains to be identified, we hypothesize it might play a role in controlling survival, growth, and/or differentiation of the dominant follicle and CL by inactivating the gonadotropin antagonist, IGFBP-4.

The effect of the angiogenesis inhibitor TNP-470 on luteal establishment and function in the primate.

Angiogenesis during luteal development is probably essential for normal lutein cell function. Since the angiogenesis inhibitor TNP-470 inhibits pregnancy in mice, the current study investigated its effects on the establishment and function of the primate corpus luteum. Regularly ovulating macaques were treated with TNP-470 (6 mg/kg), i.v. in three doses, 48 h apart. Serum progesterone concentrations, as indicators of treatment effect, were normal in four macaques where treatment commenced at the onset of the ovulatory progesterone rise, and in five of eight in which treatment commenced a few days before ovulation. In the other three the normal progesterone rise was absent. To investigate the direct effect on luteal angiogenesis of a daily dose over a longer period, four marmosets received 18 mg/kg/day of TNP-470 i.v. for 9 days starting at ovulation. On day 10, luteal cell proliferation was determined by nuclear bromodeoxyuridine incorporation. Luteal microvasculature was examined using immunocytochemical factor VIII staining, and endothelial cell and luteal function assessed by in-situ hybridization of insulin-like growth factor binding protein-3 mRNA and plasma progesterone concentrations respectively. None of these parameters were affected by the TNP-470 treatment. The results show that, with the treatment regimens employed, TNP-470 had no significant effect on the expression of the differentiated state of the primate corpus luteum.

A functional bone morphogenetic protein system in the ovary.

Bone morphogenetic proteins (BMPs) comprise a large group of polypeptides in the transforming growth factor beta superfamily with essential physiological functions in morphogenesis and organogenesis in both vertebrates and invertebrates. At present, the role of BMPs in the reproductive system of any species is poorly understood. Here, we have established the existence of a functional BMP system in the ovary, replete with ligand, receptor, and novel cellular functions. In situ hybridization histochemistry identified strong mRNA labeling for BMP-4 and -7 in the theca cells and BMP receptor types IA, IB, and II in the granulosa cells and oocytes of most follicles in ovaries of normal cycling rats. To explore the paracrine function of this BMP system, we examined the effects of recombinant BMP-4 and -7 on FSH (follicle-stimulating hormone)-induced rat granulosa cytodifferentiation in serum-free medium. Both BMP-4 and -7 regulated FSH action in positive and negative ways. Specifically, physiological concentrations of the BMPs enhanced and attenuated the stimulatory action of FSH on estradiol and progesterone production, respectively. These effects were dose- and time-dependent. Furthermore, the BMPs increased granulosa cell sensitivity to FSH. Thus, BMPs have now been identified as molecules that differentially regulate FSH-dependent estradiol and progesterone production in a way that reflects steroidogenesis during the normal estrous cycle. As such, it can be hypothesized that BMPs might be the long-sought "luteinization inhibitor" in Graafian follicles during their growth and development.

Normal reproductive and macrophage function in Pem homeobox gene-deficient mice.

Interaction between germ cells and the supporting somatic cells guides many of the differentiative processes of gametogenesis. The expression pattern of the Pem homeobox gene suggests that it may mediate specific inductive events in murine reproductive tissues. During gestation, Pem is expressed in migrating and early postmigratory primordial germ cells, as well as in all embryo-derived extraembryonic membranes. Pem expression ceases in the germline after Embryonic Day 14 in both sexes and then reappears postnatally in the supporting cells of the gonad. In mature mice, Pem is produced by testicular Sertoli cells during stages VI-VIII of spermatogenesis and transiently by ovarian granulosa cells lining periovulatory follicles. Despite this tightly regulated reproductive expression pattern, mice with a targeted mutation in Pem have normal fecundity, with no detectable alteration in extraembryonic testicular or ovarian development or function. We also show that Pem is expressed throughout embryonic and adult development in a subset of a tissue-specific class of macrophages, Kupffer cells, as well as in a localized fraction of cells in macrophage cell lines. Although the number of Pem-positive Kupffer cells increases in mice treated with lipopolysaccharide, loss of Pem does not detectably interfere with the cells' ability to induce iNOS expression, demonstrating this Kupffer cell function does not require Pem. No differences were observed between Pem-knockout mice in 129, C57BL6/J, or mixed genetic backgrounds. Together, these data show that Pem is dispensable for embryonic and postnatal development, gonadal function, and Kupffer cell activation, perhaps due to compensatory expression of a similar homeobox gene.

Insulin-like growth factor binding protein-3 mRNA expression in endothelial cells of the primate corpus luteum.

Luteinization is associated with endothelial cell proliferation as part of the extensive angiogenesis necessary to maintain corpus luteum function. However, following luteal demise, the vasculature regresses and the endothelial cells disappear. In the rat corpus luteum, the endothelial cells express high concentrations of insulin-like growth factor-binding protein-3 (IGFBP-3) during luteolysis, suggesting a role of IGFBP-3 during endothelial cell loss. The aim of the present study was to determine the occurrence and location of the messenger ribonucleic acid (mRNA) for IGFBP-3 in the primate corpus luteum, and to determine whether or not induction of luteal regression is associated with changes in localization of the message. Marmoset corpora lutea were studied throughout the cycle. The effects of induced luteolysis were examined 12 h or 24 h after treatment with either a gonadotrophin-releasing hormone antagonist or a prostaglandin F2alpha analogue, administered during the mid-luteal phase. High IGFBP-3 expression was recorded in the endothelial cells of the majority of microvessels and a minority of capillaries surrounding the lutein cells in all functionally active corpora lutea. Expression declined markedly in regressing corpora lutea of the late follicular phase. Expression of the IGFBP-3 mRNA in lutein cells in the control corpus luteum was extremely rare. There were no major differences in the degree and pattern of IGFBP-3 expression as a consequence of induced luteal regression although there was an apparent increase in the number of capillary endothelial cells expressing. Induction of luteolysis resulted in expression in a minority of lutein cells. These results support the concept that IGFBP-3 has an autocrine/paracrine role in regulating various cell types in the primate corpus luteum, including endothelial cells. However, expression of IGFBP-3 mRNA throughout the luteal phase suggests it may regulate angiogenesis and luteal function rather than endothelial cell death and luteolysis.

Evidence for a neutrophil-interleukin-8 system in human folliculogenesis.

OBJECTIVE: This study was conducted to determine whether polymorphonuclear leukocytes (neutrophils) and the potent chemoattractant interleukin-8 are associated with follicle development in the normal human ovary. STUDY DESIGN: We performed a morphometric analysis of neutrophils in 268 human ovarian follicles, of which 199 were preantral and 69 were antral. In each antral follicle the numbers of mitotic, apoptotic, and total granulosa cells were counted to determine healthy and atretic follicles. Interleukin-8 protein and messenger ribonucleic acid were detected by immunohistochemistry and in situ hybridization, respectively. RESULTS: Antral follicles contained relatively large numbers of neutrophils within the theca vasculature. The density of neutrophil was twofold greater (p < 0.05) in atretic versus healthy follicles. The neutrophil index (neutrophils/granulosa cells x 1000) was inversely correlated to the number of granulosa cells per follicle. Immunoreactive interleukin-8 was detected in the theca and granulosa cells of most all antral follicles examined. Interleukin-8 messenger ribonucleic acid was demonstrated in theca and granulosa cells of some but not all follicles examined. CONCLUSIONS: Neutrophils are present in the theca of developing antral follicles, increase in number during atresia, and are associated with expression of interleukin-8 in the follicle wall.

Activin-induced inhibin alpha-subunit production by rat granulosa cells requires endogenous insulin-like growth factor-I.

Inhibin-alpha subunit (Inh-alpha) gene expression is important for granulosa cell (GC) differentiation and prevention of ovarian tumorigenesis. Studies on Inh-alpha regulation have implicated activin and insulin-like growth factor-I (IGF-I) in the mechanisms of expression. Here we present evidence that endogenously produced IGF-I plays an obligatory role in activin-induced Inh-alpha production. Primary cultures of rat GC were incubated with increasing concentrations of various regulatory molecules, and the levels of Inh-alpha protein and its mRNA were measured in conditioned medium and cells, respectively. Recombinant activin A stimulated Inh-alpha expression, and the effects were dose- and time-dependent. The receptor tyrosine kinase inhibitor tyrphostin A23 caused a dose-dependent inhibition of activin-dependent Inh-alpha expression, whereas the inactive isomer, A63, had no effect. The stimulatory effect of activin was also blocked in a dose-dependent manner by added IGF binding protein-4 or -5, and the effects were reversed by IGF-I. Moreover, increasing concentrations of an anti-IGF-I antibody had a similar inhibitory effect on activin-stimulated Inh-alpha expression. Collectively, these results suggest, for the first time, that endogenously produced IGF-I is required for activin stimulation of Inh-alpha expression in cultured rat GC.

Endogenous insulin-like growth factor-I is obligatory for stimulation of rat inhibin alpha-subunit expression by follicle-stimulating hormone.

Insulin-like growth factor-I (IGF-I) is essential for FSH-dependent steroidogenesis by rat granulosa cells (GC), but whether IGF-I is required for other FSH-dependent functions is unknown. To investigate the role of IGF-I in the mechanisms of FSH-stimulated inhibin alpha-subunit (Inh-alpha) production, rat GC were cultured with FSH, IGF-I, insulin-like growth factor-binding protein (IGFBP)-4, IGFBP-5, and/or anti-IGF-I antibody. Inh-alpha protein and mRNA levels were measured in conditioned medium and cells by Western immunoblotting and Northern analysis, respectively. Inh-alpha expression was increased by FSH (3.5-fold) and IGF-I (2.5-fold), and the effects were dose and time dependent. FSH stimulation of Inh-alpha was attenuated by IGFBP-4 or -5 in a dose-dependent fashion, and the effects were reversed by IGF-I. Anti-IGF-I antibody mimicked the inhibitory effects of IGFBP-4 and -5. Forskolin, cholera toxin, and 8-bromo-cAMP increased Inh-alpha production approximately 3.5-fold, and the effects were blocked by IGFBP-4 or -5. Increases in Inh-alpha by FSH, IGF-I, forskolin, cholera toxin, and 8-bromo-cAMP were totally blocked by the protein tyrosine kinase inhibitor, tyrphostin A23. In summary, these results suggest that the stimulation of Inh-alpha expression by FSH requires activation of protein tyrosine kinases by endogenously produced IGF-I. We propose that the IGF-I signaling is obligatory for FSH stimulation of Inh-alpha expression in rat GC.

Defining apoptosis: players and systems.

OBJECTIVE: To summarize the basic principles and concepts of apoptosis and some of the information to date regarding the nature of the regulation of apoptosis at the molecular level. METHODS: In the past few years, research in cell and molecular biology has led to a much better understanding of the underlying mechanisms that regulate apoptosis. RESULTS: Positive and negative regulatory elements that function in specific cell types to activate apoptosis have been identified. This activation involves signaling pathways that activate distinct proteolytic cascades that lead to the execution of cell death. CONCLUSION: Homeostasis involves a delicate balance between the processes of proliferation, differentiation, and death by apoptosis. The current challenge is to understand how specific stimuli regulate the execution of programmed cell death in basic reproductive cells and how these interactions are integrated into the overall physiology and pathophysiology of reproduction during life.

Transforming growth factor-alpha stimulates insulin-like growth factor binding protein-4 (IGFBP-4) expression and blocks follicle-stimulating hormone regulation of IGFBP-4 production in rat granulosa cells.

The ability of TGF-alpha to regulate insulin-like growth factor binding protein-4 (IGFBP-4), was investigated. Primary cultures of rat granulosa cells (GC) were grown in serum-free medium with rat (r) TGF-alpha and/or rFSH, and secreted IGFBP-4 protein and its steady state mRNA levels were measured by Western immunoblotting and Northern blotting, respectively. Control (untreated) cells secreted IGFBP-4 spontaneously, and the levels were increased by rTGF-alpha in a dose- and time-dependent manner. rTGF-alpha abolished FSH-induced IGFBP-4 protease activity and suppressed FSH-dependent effects on IGFBP-4 production. IGFBP-4 mRNA levels were decreased and increased by FSH and TGF-alpha, respectively, and TGF-alpha blocked the FSH effects. These results demonstrate that TGF-alpha is a potent stimulator of IGFBP-4 expression in rat GC and can overcome the regulatory effects of FSH on IGFBP-4 production. The consequence of these TGF-alpha effects is a marked, sustained increase in the levels of IGFBP-4 in the microenvironment.

Physiologic basis of ovulation induction.

Optimal regimens for ovulation induction depend on a sound, fundamental understanding of the physiology of ovulation. This chapter will review the endocrinology of folliculogenesis, with particular emphasis on the signaling pathways activated by FSH and LH. Important new information regarding the molecular mechanisms of signal transduction and the biologic responses that are produced will also be presented. Ultimately, a clearer understanding of mechanisms regulating the selection and promotion of the dominant follicle, as described here, will lead to advances in the field of ovulation induction and pregnancy in women.

Activin causes premature superovulation.

In the rat ovary, follicle cells produce and respond to activin, but as yet, the functional significance of the autocrine/paracrine effects of ovarian activin remains equivocal. To assess the effects of activin on folliculogenesis, normal cycling female rats were injected once every 8 h over a 40-h period with recombinant human activin A (120 micrograms/kg) beginning at 1300 h on estrus (day 1 of treatment). A total of 10 rats were injected with activin in 2 separate experiments. On days 3 and 4 of treatment, blood was obtained for hormone measurements, and the ovaries were removed for histology. Follicle counts were performed in 1 ovary from 3 representative animals in each treatment group. All antral (Graafian) follicles 300 microns or more in diameter were measured and classified as healthy or atretic based on the number of pyknotic nuclei in the largest cross-section. On day 3, all rats were in diestrus (diestrous day 2). After 3 days of activin administration, serum levels of estradiol were increased 200%, progesterone levels were decreased 67%, and FSH levels were unchanged compared with those in matched controls. By day 4 (i.e. 1 day after the last injection), no changes in the levels of these hormones were observed. Injection of activin for 3 days did not change the total number of antral follicles per ovary (control, 41.3 +/- 4.9; activin, 43.7 +/- 3.9); however, activin significantly increased the total number of atretic follicles (control, 69%; activin, 92%). Morphometric analysis of the ovaries removed on day 3 showed a marked increase (2-fold) in the number of large follicles, but most (89%) were atretic. Follicle counts suggested that the additional large follicles may have come from the pool of healthy small follicles. Histological studies showed that some of the day 3 activin-treated follicles had initiated ovulation. On day 4, control and activin-treated animals were at proestrus and estrus, respectively. Therefore, activin shortened the estrous cycle by 1 day. Little or n change in the follicle populations was observed in day 4 control ovaries. Interestingly, however, in 2 of the 3 activin-treated animals, 1 set of large follicles had ovulated (12 +/- 1 expanded egg cumulus complexes/oviduct), and another set (13 +/- 2) was just about to rupture. The activin-exposed oocytes (tubal and follicular) appeared arrested in metaphase I. After ovulation, activin-treated follicles developed into typical corpora lutea. No ovulations were found in control animals.(ABSTRACT TRUNCATED AT 400 WORDS)