Bone morphogenetic proteins (BMPs) comprise a large group of polypeptides in the transforming growth factor ␤ 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 (folliclestimulating 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 timedependent. 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 ref lects 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.

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.

Summary. The concentrations of progesterone, androstenedione, testosterone, oestrone and oestradiol were measured by radioimmunoassay in blastocysts and uterine fluid flushings collected from rabbits 110 to 159 h post coitum (p.c.). None of the blastocysts or uterine flushings contained detectable levels of androstenedione, testosterone or oestrone. All uterine flushings contained large amounts of progesterone and some of the flushings also contained oestradiol.A small amount of progesterone (\ m=~\ 7\ m=. \ 5pg/blastocyst) was first detectable in some blastocysts at 135 h p.c.; progesterone levels/blastocyst then increased progressively, reaching levels of about 122\p=n-\158 pg/blastocyst at 159 h p.c. Micropuncture of blastocysts at 159 h p.c. indicated that \ m=ge\ 90 % of the progesterone in the embryo was in the blastocoelic fluid. Blastocysts from rabbit uterine horns containing oestradiol also contained oestradiol but those in which oestradiol was detected were never observed in uteri lacking the hormone. It is inferred that rabbit blastocysts accumulate both progesterone and oestradiol from uterine fluid.

There is evidence that insulin-like growth factor binding protein-3 (IGFBP-3) is a part of the intrinsic IGF system in rat CL. Here, we examined when during luteogenesis the IGFBP-3 gene is expressed. IGFBP-3 messenger RNA (mRNA) was characterized by Northern analysis and in situ hybridization techniques. Animals were selected at proestrus (P 1000 h and P 2000 h), estrus (E 0200 h and E 1000 h), diestrus I (DI 1100 h), and diestrus II (DII 1100 h), and in pregnancy (day 12, before luteolysis and day 21, during luteolysis). A single 2.6 kilobase IGFBP-3 transcript was identified at each stage of the estrous cycle; however, the amounts of message varied markedly, being most abundant at P 1000 h, least abundant at P 2000 h, E 0200 h, E 1000 h, and DI, and then more abundant at DII. Corroborating our earlier report, IGFBP-3 mRNA was limited solely to corpora lutea (CL). Newly-formed CL-I at E 0200 h and E 1000 h revealed no IGFBP-3 hybridization. This is the period of early luteinization when cells undergo hypertrophy and capillaries and lymphatics penetrate the granulosa lutein layer. At DI 1100 h, a few cells (12.2 +/- 3.4%) near the central cavity of the CL-I showed a positive hybridization signal for IGFBP-3; this period is commensurate with active luteinization when the vascular tissue develops a distinctly sinusoidal character and progesterone secretion by CL-I increases. At DII 1100 h, more cells in the central area were positive for IGFBP-3 (55.2 +/- 6.4%); this is the period of active luteolysis when P4 secretion has fallen to basal levels. At P 1000 h, a positive IGFBP-3 hybridization signal was detected in most CL-I cells (85.3 +/- 2.8%), and the signal was particularly intense in subtypes of endothelial cells lining venous sinusoids and/or lymphatics and some perivascular cells; this is the period when patches of pyknotic cells appear in the central area of CL-I. At P 2000 h, 45.9 +/- 1.6% of the CL-I cells showed a positive signal; however, the intensity of the signal was much weaker when compared to P 1000 h. During the next cycle, the CL-I become the CL of the second generation (CL-II), which show increased necrosis. Between estrus and diestrus II of the next cycle, a large number of the CL-II cells (approximately 75%) were positive for IGFBP-3 and the signal was very strong in some groups of cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Summary. Rabbit oocytes from large (1\ p=n-\ 1\ m=. \ 5mm diam.), medium (0\m=.\5mm) and small (0\m=.\15\p=n-\0\m=.\25mm) antral follicles were cultured in five chemically defined media. In all media, oocytes from large antral follicles showed the highest incidence of meiotic activity followed by those from follicles of medium size. Most oocytes from small follicles did not resume meiosis in culture. The addition of glutamine to a standard medium for ovum culture significantly improved maturation of oocytes from medium\x=req-\ sized follicles but did not affect those from large or small follicles. When polyvinylpyrrolidone was substituted for bovine serum albumin, maturation of oocytes from large and medium-sized follicles was reduced. Progesterone at a concentration of 10 \g=m\M did not affect maturation, but 100 \g=m\M-progesteroneblocked germinal vesicle breakdown in oocytes from medium-sized follicles and reduced both germinal vesicle breakdown and polar body formation in oocytes from large follicles. This effect was reversible.