To determine the physiological significance of tumor necrosis factor α (TNFα) in the regulation of luteolytic prostaglandin (PG) F_2α release by the bovine endometrium, the effect of TNF-α on PGF_2α output by the endometrial tissues in vitro was investigated and compared with the effect of oxytocin (OT). Furthermore, the presence of specific receptors for TNFα in the bovine endometrium during the estrous cycle was determined. Endometrial slices (20–30 mg) taken from six stages of the estrous cycle (estrus: Day 0; early I: Days 2–3; early II: Days 5–6; mid-: Days 8–12; late: Days 15–17; and follicular: Days 19–21), as determined by macroscopic examination of the ovaries and uterus, were exposed to TNFα (0.06–6 nM) and/or OT (100 nM). OT stimulated PGF_2α output at the follicular stage and at estrus (P < 0.001), but not at the late luteal stage. On the other hand, the stimulatory effects of TNFα on PGF_2α output were observed not only at the follicular stage but also at the late luteal stage (P < 0.001). When the endometrial tissues at late luteal stage were simultaneously exposed to TNFα (0.6 nM) and OT (100 nM), the stimulatory effect on PGF_2α output was higher than the effect of TNFα or OT alone (P < 0.05). Specific binding of TNFα to the bovine endometrial membranes was observed throughout the estrous cycle. The concentration of TNF-α receptor at the early I luteal stage was less than the concentrations at other luteal stages (P < 0.01). The dissociation constant (K_d) values of the endometrial membranes were constant during the estrous cycle. The overall results lead us to hypothesize that TNFα may be a trigger for the output of PGF_2α by the endometrium at the initiation of luteolysis in cattle.

Tumor necrosis factor α (TNFα) has been shown to be a potent stimulator of prostaglandin (PG) F_2α secretion in the bovine endometrium. The aims of the present study were to determine the cell types in the endometrium (epithelial or stromal cells) responsible for the secretion of PGF_2α in response to TNFα, and the intracellular mechanisms of TNFα action. Cultured bovine epithelial and stromal cells were exposed to TNFα (0.006–6 nM) or oxytocin (100 nM) for 4 h. TNFα resulted in a dose-dependent increase of PGF_2α production in the stromal cells (P < 0.001) but not in the epithelial cells. On the other hand, oxytocin stimulated PGF_2α output in the epithelial cells but not in the stromal cells. When the stromal cells were incubated for 24 h with TNFα and inhibitors of phospholipase (PL) C or PLA₂, only PLA₂ inhibitor completely stopped the actions of TNFα (P < 0.001). When the stromal cells were exposed to TNFα and arachidonic acid, the action of TNFα was augmented (P < 0.001). When the stromal cells were incubated for 24 h with a nitric oxide (NO) donor (S-NAP), S-NAP stimulated the PGF_2α production dose-dependently. Although an NO synthase (NOS) inhibitor (l-NAME) reduced TNFα-stimulated PGF_2α production, an inhibitor of phosphodiesterase augmented the actions of TNFα and S-NAP (P < 0.05). The overall results indicate that the target of TNFα for stimulation of PGF_2α production in cattle is the endometrial stromal cells, and that the actions of TNFα are mediated via the activation of PLA₂ and arachidonic acid conversion. Moreover, TNFα may exert a stimulatory effect on PGF_2α production via the induction of NOS and the subsequent NO-cGMP formation.

We hypothesize that spontaneous regression of corpora lutea (CL) involves short-lasting restructure of luteal tissue with an activation of matrix metalloproteinases (MMPs) and their respective inhibitors (tissue inhibitors of metalloproteinase, TIMPs). This was tested by determining the gene expression of MMP-1, MMP-2, and MMP-9 and respective TIMP-1 and TIMP-2 in luteal tissue from sows at the early, midluteal, and late luteal phase (Days 6–8, Days 9–11, and Days 13–15 of estrous cycle). Gene expression of the three MMPs was low in early, slightly higher in midluteal, and significantly elevated (P < 0.05) in regressing CL. An inverse pattern was found for gene expression of TIMP-1 and TIMP-2. Under culture conditions, the release of MMPs was determined from steroidogenic large luteal cells (LLC). LLC harvested from regressing CL released significantly (P < 0.05) more active MMPs than cells obtained from CL at the early luteal phase. As luteolysis can be induced by prostaglandin F_2α (PGF_2α) and tumor necrosis factor α (TNF), we studied their effects on LLC under culture conditions. Treatment of cells with PGF_2α or TNF (10⁻⁷ M or 3 × 10⁻⁹ M, respectively) induced a significantly higher release of MMPs, and gene expression was also significantly stimulated in comparison to that in untreated LLC. The gene expression of TIMPs remained unaffected by either treatment. It is concluded that at the beginning of luteolysis, MMPs are expressed and released in high amounts and that this is essential for the structural regression of the CL.

Inhibins and activins are dimeric peptide hormones that play an integral role in the intraovarian regulation of folliculogenesis. The domestic hen, with its well-defined follicular hierarchy, provides a unique model in which to study the role of these hormones in follicular development. In the present study, the complete coding sequence and deduced amino acid sequence for the chicken inhibin/activin β_B subunit has been determined from cDNA clones isolated from a chicken ovarian granulosa cell library. This β_B-subunit cDNA predicts a precursor protein of 392 amino acids containing the mature C-terminal 115 amino acid β_B subunit. When compared to the β_B subunit isolated from a variety of species, the chicken cDNA clone showed high nucleotide identity in the full-length coding region (>70%) and in the mature coding region (>80%). In addition, the deduced amino acid sequence of chicken β_B subunit showed greater than 95% identity compared to other species in the mature peptide region. Expression of the β_B-subunit mRNA was detected by reverse transcription-polymerase chain reaction in both gonadal and extragonadal tissues. Northern blot analysis detected expression in the gonadal tissues only, specifically in the granulosa tissue from the F3-F5 follicles, small yellow follicles (SYF), large white follicles, and immature and mature rooster testes. A major transcript of approximately 4.1 kilobases (kb) and three minor transcripts of approximately 8.4 kb, 6.5 kb, and 1.7 kb were detected in the SYF granulosa samples. To examine the expression pattern of the β_B subunit around the stage of follicle selection, the SYF granulosa was subdivided into two groups: 6–8 mm and 9–12 mm. Quantification of RNA expression (n = 3) showed that expression of the β_B subunit was maximal in the 6–8 mm SYF. Activin B, as well as other intraovarian signals, may regulate early follicle selection and/or development in the chicken.

This study compared the nuclear transfer (NT) embryo development rates of adult and fetal cells within the same genotype. The adult fibroblast cells were obtained from a 21-yr-old Brahman bull. The fetal cells were derived from a Day 40 NT fetus previously cloned using cells from the Brahman bull. Overall, similar numbers of blastocysts developed from both adult (53 of 190; 28%) and fetal (39 of 140; 28%) donor cells. Improved blastocyst development rates were observed when fetal cells were serum-starved (serum-fed 12% vs. serum-starved 43%; P < 0.01) whereas there was no similar benefit when adult cells were serum-starved (both serum-fed and serum-starved 28%).

Day 30 pregnancy rates were similar for blastocysts derived from adult (6 of 26; 23%) or fetal (5 of 32; 16%) cells. Day 90 pregnancy rates were 3 of 26 for adult and 0 of 32 for the fetal cell lines. One viable bull calf derived from a 21-yr-old serum-starved adult skin fibroblast was born in August 1999. In summary, somatic NT embryo development rates were similar whether adult or fetal cells, from the same genotype, were used as donor cells. Serum starvation of these adult donor cells did not improve development rates of NT embryos to blastocyst, but when fetal cells were serum-starved, there was a significant increase in development to blastocyst.

Estrogen production by the preimplantation equine embryo is presumed to be important in maternal-conceptus communication in the mare. The synthesis of C₁₈ estrogens from C₁₉ androgens requires cytochrome P450 aromatase (P450_arom) in the conceptus, but little information is available on the specific tissue location or potential developmental patterns of expression for the horse. The goal of this research was to localize P450_arom in the equine conceptus by immunocytochemistry and in situ hybridization. Intact blastocyst-stage embryos were collected by nonsurgical flush on Days 12–15 of pregnancy, fixed in 4% paraformaldehyde, and paraffin-embedded. Aromatase protein was localized using rabbit anti-human placental aromatase antiserum with a detection system utilizing peroxidase and 3-amino-9-ethylcarbazole. For in situ hybridization, tissue sections were incubated with sense or antisense [³⁵S]UTP-labeled cRNA probes prepared from equine aromatase cDNA. Aromatase protein and transcript were abundant in the extraembryonic trophectoderm but absent from embryonic ectoderm. No P450_arom expression was detected in abembryonic endoderm or mesoderm. Aromatase expression was demonstrated in the endoderm beneath the disc (hypoblast). This pattern of P450_arom expression in the equine blastocyst closely resembles that seen transiently in the porcine embryo, suggesting that regulatory mechanisms conferring tissue specificity may be conserved.

Sertoli cells express functional receptors for FSH, one of the two pituitary hormones that regulate spermatogenesis in mammals. We recently produced genetic mutant (FORKO) mice that lack FSH receptor, in order to examine the effects on testicular function and fertility. Mutant males exhibited weight loss of testis, epididymis, and seminal vesicle as well as low levels of testosterone. Except for reduced seminiferous tubular diameter, no gross changes were apparent upon histological examination. Analysis of testicular germ cells by flow cytometry revealed a significant increase in the percentage of 2C cells (spermatogonia and non-germ cells) and a significant decrease in the percentage of HC cells (elongated spermatids) of FORKO males. The absolute number of homogenization-resistant elongated spermatids was also significantly reduced in the mutant males. A 2-fold increase in c-kit-positive 2C cells was recorded in the mutant males. Elongated spermatids of FORKO males showed a dramatic increase in propidium iodide binding suggesting reduced nuclear compaction. The increase in size of the sperm head in mutants, as well as susceptibility to dithiothreitol-induced decondensation, suggests the inadequate condensation of sperm chromatin. Sperm chromatin structure assay, a technique that reflects DNA stability, revealed that sperm from FORKO males are susceptible to acid denaturation, indicating the poor quality of sperm. These data allow us to conclude that genetic disruption of FSH receptor signaling in the rodent induces major changes that might contribute to reduced fertility.

As a step in elucidating the biological role of plasma kallikrein (PK) present in the follicular fluid of mammalian ovaries, we examined pig ovary fluid to determine its constituent activators and substrates. Using the inactive precursor form of plasma kallikrein (prePK) as a substrate, we purified an enzyme capable of activating this protein. The prePK-activating enzyme was shown to be the active enzyme blood coagulation factor XIIa. We also isolated high molecular weight kininogen (HMW-K) from the same fluid. Incubation of HMW-K with the ovarian follicular fluid PK resulted in the production of the nanopeptide bradykinin (BK). Expression of prePK, blood coagulation factor XII, and HMW-K was examined by Northern blot analysis using ovary and liver poly(A) RNA. All these transcripts were found in the liver, but none were found in the ovary. In addition, it was found that BK levels in the fluid derived from the small follicles were approximately 6 times higher than those from medium and large follicles. These results demonstrate the presence of a BK-producing system in the ovarian follicles and suggest the physiological importance of this peptide hormone in the early stages of follicular development and at ovulation.

Endometrial receptivity for implantation and sensitization for decidualization in rodents is a transient state under the control of the ovarian steroids estrogen and progesterone. It is unclear, however, what molecular events mediate the onset of uterine receptivity. Messenger RNA differential display was performed on endometrial RNA from ovariectomized rats differentially sensitized for decidualization. Maximally sensitized uteri were at the equivalent of Day 5 of pseudopregnancy, and temporally nonsensitized uteri at Day 4 or 6; hormonally nonsensitized uteri were from animals on Day 5 treated with low or high doses of estradiol on Day 4. A cDNA with endometrial expression restricted to maximally sensitized uteri was isolated, cloned, and sequenced. The cDNA matched the sequence for glucose-regulated protein 78 (GRP78), a heat shock 70-related protein that resides in the lumen of the endoplasmic reticulum (ER) and has roles in several cellular processes including multimeric protein assembly, the degradation of proteins, and the storage and regulation of ER luminal calcium. Northern blot analysis indicated a dramatic increase in GRP78 mRNA levels restricted to the sensitized, Day 5 endometrium, suggesting a role in the onset of the sensitized phase. In situ hybridization and immunohistochemistry experiments localized the up-regulation of GRP78 within the receptive endometrium to the glandular epithelium.

Actin is one of the major proteins in mammalian oocytes. Most developmental events are dependent on the normal distribution of filamentous (F-) actin. Polymerization of nonfilamentous (G-) actin into F-actin is important for both meiosis and mitosis. This study examined G- and F-actin distribution in pig oocytes and embryos by immunocytochemical staining and confocal microscopy. Actin protein was quantified by electrophoresis and immunoblotting. G-Actin was distributed in the whole cytoplasm of oocytes and embryos irrespective of their stages. F-Actin was distributed at the cortex of oocytes and embryos at all stages, at the joint of blastomeres in the embryos, in the cytoplasm around the germinal vesicle (GV), and in the perinuclear area of 2- to 4-cell-stage embryos. No differences in the amount of actin protein were found among oocytes and embryos. Oocytes cultured in medium with cytochalasin D (CD), an inhibitor of microfilament polymerization, underwent GV breakdown and reached metaphase I but did not proceed to metaphase II. Two- to 4-cell-stage embryos cultured in medium with CD did not develop to blastocysts. When GV-stage oocytes or 2- to 4-cell-stage embryos treated with CD for 6 h were re-cultured in media without CD, oocytes or embryos re-assembled actin filaments and underwent a meiotic maturation or blastocyst formation similar to that of controls. These results indicate that it is the polymerization of G-actin into F-actin, not actin protein synthesis, that is important for both meiosis and mitosis in pig oocytes and embryos.

This work focuses on the assembly and transformation of the spindle during the progression through the meiotic cell cycle. For this purpose, immunofluorescent confocal microscopy was used in comparative studies to determine the spatial distribution of α- and γ-tubulin and nuclear mitotic apparatus protein (NuMA) from late G2 to the end of M phase in both meiosis and mitosis. In pig endothelial cells, consistent with previous reports, γ-tubulin was localized at the centrosomes in both interphase and M phase, and NuMA was localized in the interphase nucleus and at mitotic spindle poles. During meiotic progression in pig oocytes, γ-tubulin and NuMA were initially detected in a uniform distribution across the nucleus. In early diakinesis and just before germinal vesicle breakdown, microtubules were first detected around the periphery of the germinal vesicle and cell cortex. At late diakinesis, a mass of multi-arrayed microtubules was formed around chromosomes. In parallel, NuMA localization changed from an amorphous to a highly aggregated form in the vicinity of the chromosomes, but γ-tubulin localization remained in an amorphous form surrounding the chromosomes. Then the NuMA foci moved away from the condensed chromosomes and aligned at both poles of a barrel-shaped metaphase I spindle while γ-tubulin was localized along the spindle microtubules, suggesting that pig meiotic spindle poles are formed by the bundling of microtubules at the minus ends by NuMA. Interestingly, in mouse oocytes, the meiotic spindle pole was composed of several γ-tubulin foci rather than NuMA. Further, nocodazole, an inhibitor of microtubule polymerization, induced disappearance of the pole staining of NuMA in pig metaphase II oocytes, whereas the mouse meiotic spindle pole has been reported to be resistant to the treatment. These results suggest that the nature of the meiotic spindle differs between species. The axis of the pig meiotic spindle rotated from a perpendicular to a parallel position relative to the cell surface during telophase I. Further, in contrast to the stable localization of NuMA and γ-tubulin at the spindle poles in mitosis, NuMA and γ-tubulin became relocalized to the spindle midzone during anaphase I and telophase I in pig oocytes. We postulate that in the centrosome-free meiotic spindle, NuMA aggregates the spindle microtubules at the midzone during anaphase and telophase and that the polarity of meiotic spindle microtubules might become inverted during spindle elongation.

The present study tested the hypothesis that macrophage distribution and activation are enhanced in the uterus before term. Mid-uterine horn tissue strips from mice on Days 15 and 18 of pregnancy, the day of birth (= Day 19), and one day postpartum were paraffin-embedded and then sectioned, stained with a monoclonal pan-macrophage marker (BM8), and processed for visualization and quantification of resident macrophages per nuclear area. Macrophages were dispersed throughout the endometrium and subluminal epithelium; cell numbers declined on the day before term, then increased postpartum. Within myometrium, macrophages congregated in stroma surrounding muscle bundles, and staining was enhanced near term. Macrophage numbers were similar in pregnant and postpartum uteri, enhanced more than 2-fold over those in nonpregnant controls. Uterine sections were also analyzed by laser-scanning cytometry to enumerate activated macrophages (i.e., those that express the intercellular adhesion molecule marker CD54 ) and to determine cell cycle (propidium iodide fluorescence). Activated macrophages were directly proportional to cell numbers and, by cell cycle analysis, were not terminally differentiated. Highest cell numbers occurred on Day 15: 4-fold greater than those in nonpregnant controls and 2-fold higher than those at Day 18 or in postpartum groups. These findings indicate a decline in endometrial macrophage numbers at least one day before the onset of parturition and raise the possibility that trafficking of this immune cell may contribute to onset of labor.

SP-10 is a sperm intra-acrosomal protein, specific to the testis, that is believed to play an important role in egg-sperm binding. While the molecular characterization of the SP-10 protein has been clarified, little is yet known of its functional role in fertilization. We therefore established a monoclonal antibody (mAb pep-SP10) against a peptide (pep-SP10) that included the most hydrophilic portion of human SP-10 between the 135th and 149th amino acids. Human SP-10 was found to be localized in the equatorial region of acrosome-reacted sperm by immunofluorescent staining using our mAb pep-SP10. Monoclonal Ab pep-SP10 inhibited sperm-oolemma binding in the zona-free hamster egg penetration test, but it did not inhibit sperm-zona binding in the hemizona assay. Furthermore, we demonstrated that the oolemmal ligands of human SP-10 did not include β₁ integrins, the most promising candidates for oocyte ligands involved in sperm-oolemma binding, based on the findings of a human sperm-cultured cell binding assay using F9 mouse embryonal carcinoma cells and F9-transformed cells lacking β₁ integrins. In conclusion, our present data suggest that human SP-10, expressed on the equatorial region of acrosome-reacted sperm, indeed mediates sperm-oolemma binding in a β₁ integrin-independent manner, but not sperm-zona binding.

The first objective of this study was to determine whether the death of bovine granulosa cells (GC) isolated from small (≤ 4 mm), medium (5–8 mm), and large (> 8 mm) follicles during follicular atresia occurs by apoptosis. The second objective was to establish an in vitro model system to elucidate the developmental (GC from follicles of different sizes) and hormonal (FSH and insulin-like growth factor-I [IGF-I]) regulation of bovine GC apoptosis during follicular atresia. Bovine ovaries were obtained from a nearby slaughterhouse. Follicles were classified by morphometric criteria as healthy or atretic. Apoptosis in GC from follicles of different sizes was analyzed by both morphological and biochemical methods. Bovine GC were cultured for 48 h at a density of 5 × 10⁶ cells/ml in serum-free media at 39°C to determine the effects of FSH and IGF-I on apoptosis. The results showed that apoptosis occurred in GC from all sizes of follicles. Apoptosis in GC was also detected in some healthy follicles. Degenerate GC displayed the morphological characteristics of apoptosis, including nuclei with marginated chromatin, a single condensed nucleus, multiple nuclear fragments, and/or membrane-bound structures containing variable amounts of chromatin and/or cytoplasm (apoptotic bodies). All GC classified as apoptotic on the basis of their morphology contained fragmented DNA measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique. Cells that had undergone apoptosis were observed mainly in GC and in scattered theca cells. Throughout the GC layer, apoptotic cell death was more prevalent among antral GC than among mural GC. Interestingly, morphological results showed that no apoptosis occurred in cumulus cells. A time-dependent, spontaneous onset of apoptosis occurred in GC from small, medium, and large follicles during in vitro serum-free culture. The rate of DNA fragmentation in the culture of GC from small follicles was higher than that from medium and large follicles. FSH attenuated apoptotic cell death in GC from medium follicles more effectively than in those from small follicles. IGF-I also suppressed apoptosis in cultured GC from small follicles. In conclusion, this study showed that 1) GC death during bovine follicular development and atresia occurs by apoptosis; 2) apoptosis occurs in GC and theca cells; however, apoptosis does not occur in cumulus cells even in atretic antral follicles; 3) GC from all small, medium, and large follicles undergo spontaneous onset of apoptosis when cultured under serum-free conditions; and 4) FSH and IGF-I can attenuate apoptosis in cultured bovine GC.

Cryopreservation of ovarian cortical tissue and subsequent transplantation or in vitro culture of follicles are technologies under development with the aim to safeguard fertility in patients with gonadal failure. In the present study, we investigated whether primordial follicles could be triggered to full maturation by a combination of in vivo transplantation and in vitro culture in a mouse model. In a first step, newborn mouse ovaries containing only primordial follicles were allotransplanted under the renal capsule of ovariectomized recipient mice. The second step was to mechanically isolate growing preantral follicles from the graft and culture these in vitro to maturity. In our experiment, one newborn mouse ovary was transplanted under the renal capsule of each 8- to 12-wk-old F1 (C57Bl/6j × CBA/Ca) female ovariectomized recipient (n = 26). Two weeks after transplantation, all 26 grafts were recovered. Four grafts were processed for histology and showed that developmental stages of follicles in 14-day-old ovarian grafts were comparable to those in 14-day-old mouse ovaries. The 22 remaining grafts were used for mechanical isolation of preantral follicles. As a control group, preantral follicles isolated from ovaries of 14-day-old mice were used. The mean preantral follicle yield per ovary was 11 in the transplant group versus 33 in the control group. Follicles were cultured individually in 20-μl droplets of α-MEM supplemented with 100 mIU rFSH and 5% fetal bovine serum for 12 days under an atmosphere of 5% CO₂ in air at 37°C. By Day 12 of culture, 66.5% of follicles retained their oocytes in the grafting group versus 97.5% in the control group (P < 0.001). Final oocyte maturation was induced by addition of 2.5 IU/ml hCG. At 14–16 h post-HCG, the percentages of oocytes showing germinal vesicle breakdown and polar body extrusion were significantly higher in the control group (90.6% and 82.8%) compared to the grafting group (60% and 45%). The mean diameter of the mature oocytes of the grafting group (69.9 ± 4.45 μm) was similar to that of oocytes from the control group (70.5 ± 2.35 μm). Our results suggest that maturation of mouse primordial follicles is feasible by combination of in vivo transplantation and in vitro culture. This two-step strategy may be an attractive model for promoting the growth and maturation of primordial follicles from other species.

Activin A has been shown to exert several regulatory functions on human placenta. In the present study, we tested the hypothesis that activin A is an autocrine regulator of trophoblast using a choriocarcinoma cell line, JEG-3, as a model. Messenger RNAs for activin β_A subunit, activin binding protein (follistatin), and various activin receptors, including ActR-IA, ActR-IB, ActR-IIA, and ActR-IIB, were detected in JEG-3 cells by reverse transcription-polymerase chain reaction. The expression of activin A in JEG-3 cells was further confirmed by Western blot analysis using an antibody against activin β_A subunit. Using Northern blot analysis, Smad-2 and Smad-4 mRNAs were also observed in JEG-3 cells. These data suggest that JEG-3 cells produce activin A and express activin binding proteins and receptors, as well as potential downstream signals. In cultured JEG-3 cells, basal progesterone production was stimulated by activin A but inhibited by follistatin-288. Similarly, in the presence of androstenedione, estradiol production was enhanced by activin A but decreased by follistatin-288. On the other hand, neither activin A nor follistatin affected JEG-3 cell growth. Taken together, these findings strongly suggest that activin A is an autocrine factor that is involved in the regulation of progesterone and estradiol production in JEG-3 cells.

Although Ca² is of fundamental importance in mammalian sperm capacitation, its downstream targets have not been definitively demonstrated. The purpose of this study was to use the calmodulin (CaM) antagonists W7 and calmidazolium (CZ) to investigate the possible role of CaM, a Ca² -specific binding protein, in capacitation. Sperm membrane changes associated with capacitation were assessed by the B pattern after chlortetracycline staining and by the ability to undergo the acrosome reaction (AR) in response to lysophosphatidylcholine (LPC). The percentage of B pattern sperm was significantly inhibited by W7 or CZ in a concentration-dependent manner. At 100 μM W7 or 10 μM CZ, these inhibitors also significantly reduced the sperm's ability to undergo the LPC-induced AR. Inhibition of the B pattern and the LPC-induced AR was overcome by exogenous cAMP analogues. Treatment of the sperm with 100 μM W7 also resulted in a significant decrease in their ability to fertilize eggs in vitro. At 100 μM, W5, a less potent dechlorinated W7 analogue, had no effect on the B pattern, LPC-induced AR, or fertilization competence. Sperm viability and protein tyrosine phosphorylation were not substantially affected by 100 μM W7 (relative to 100 μM W5) or 10 μM CZ; however, the percentages of motile and hyperactivated sperm were significantly reduced. The antagonist-inhibited sperm motility was restored by dilution in control medium, but not by cAMP analogues. These results suggest that CaM participates in the regulation of membrane changes important for mouse sperm capacitation, at a point upstream from cAMP, and that this pathway is at least partially separable from pathways controlling tyrosine phosphorylation and hyperactivation.

The rat FSH receptor (rFSHR) shares considerable homology with the rat LH receptor (rLHR), yet binds human FSH (hFSH) with high fidelity, suggesting that the binding determinant encoded by the rFSHR gene shares no homology with the analogous rLHR primary sequence, thereby affording specificity of ligand binding. Two such regions of primary sequence have been previously identified and studied by peptide challenge tests and immunoneutralization studies. We therefore implemented site-directed mutagenesis to delete the regions S9-N30 and D300-F315 of the mature rFSHR sequence. The mutant receptor (ΔrFSHR) cDNAs were expressed in insect cells. The large deletion ΔrFSHRS9-N30 and a smaller deletion, ΔrFSHRS9-S18, did not bind ¹²⁵I-hFSH. However, ΔrFSHRK19-R29 and ΔrFSHRD300-F315 bound ¹²⁵I-hFSH with an affinity indistinguishable from wild-type rFSHR. The deletion mutants ΔrFSHR S9-N30 or ΔrFSHRS9-S18 were not detectable on the cell surface by flow cytometry unless cells were sheared. Although ¹²⁵I-hFSH binding to ΔrFSHRK19-R29 was normal, this form of the receptor was defective for signal transduction whereas ΔrFSHRD300-F315 was not. Furthermore, neither region seems to be a specificity determinant, since their removal did not result in high-affinity binding of hCG to ΔrFSHR.

This study tested the hypothesis that LH secretion is modulated by insulin and that the responsiveness to hypoinsulinemia is enhanced by sex steroids. The model was the developing male lamb (12–26 wk of age) rendered diabetic by chemically induced necrosis of insulin-secreting tissue (streptozotocin). Our approach was to monitor LH secretion under diabetic conditions, with or without insulin supplementation, either in the presence or in the absence of gonadal steroids. The first experiment determined if chronic insulin supplementation could sustain LH secretion in diabetic lambs. After documentation of the induced diabetic condition, twice-daily treatment with a long-acting insulin preparation (Lente) minimized diabetes-induced hyperglycemia, sustained growth, and maintained LH pulse frequency at levels comparable to pre-diabetic conditions. A second experiment evaluated the acute regulation of LH secretion by insulin. Twenty-four hours of insulin withdrawal decreased LH pulse frequency, increased circulating glucose levels, increased the concentration of plasma non-esterified fatty acids (NEFAs), and increased urinary output of ketones. LH pulse frequency continued to decline after 96 h of insulin withdrawal. By contrast, 24 h of insulin re-supplementation increased LH pulse frequency, reduced circulating glucose and NEFA concentrations, decreased plasma cortisol, and reduced urinary output of ketones. After 96 h of insulin re-supplementation, LH pulse frequency increased further, to levels comparable with those before insulin withdrawal. A third experiment determined if the effects of insulin withdrawal on LH secretion are influenced by the presence of gonadal steroids. The same individuals were treated with a physiologic dose of estradiol (Silastic capsule, s.c.) and subsequently monitored for changes in LH secretion in the presence and in the absence of exogenous insulin. Prior to insulin withdrawal, estradiol decreased both LH pulse frequency and pulse amplitude. Moreover, after 96 h of insulin withdrawal, estradiol potentiated the decline in LH pulse frequency (47% reduction in LH pulse frequency in the presence of estradiol versus 26% reduction in LH pulse frequency in the absence of estradiol). These findings support the contention that insulin and/or insulin-dependent changes in glucose availability modulate LH(GnRH) pulse frequency, and that such effects are potentiated by, but not dependent upon, gonadal steroids.

This study tested the hypothesis that central mechanisms regulating luteinizing hormone (LH) secretion are responsive to insulin. Our approach was to infuse insulin into the lateral ventricle of six streptozotocin-induced diabetic sheep in an amount that is normally present in the CSF when LH secretion is maintained by peripheral insulin administration. In the first experiment, we monitored cerebrospinal fluid (CSF) insulin concentrations every 3–5 h in four diabetic sheep given insulin by peripheral injection (30 IU). The insulin concentration in the CSF was increased after insulin injection, and there was a positive relationship between CSF and plasma concentrations of insulin (r = 0.80, P < 0.01). In the second experiment, peripheral insulin administration was discontinued, and the sheep received either an intracerebroventricular (i.c.v.) infusion of insulin (12 mU/day in 2.4 ml saline) or saline (2.4 ml/day) for 5 days (n = 6) in a crossover design. The dose of insulin (i.c.v.) was calculated to approximate the increase in CSF insulin concentration found after peripheral insulin treatment. To monitor LH secretory patterns, blood samples were collected by jugular venipuncture at 10-min intervals for 4 h on the day before and 5 days after the start of i.c.v. insulin infusion. To monitor the increase in CSF insulin concentrations, a single CSF sample was collected one and four days after the start of the central infusion. The i.c.v. insulin infusion increased CSF insulin concentrations above those in saline-treated animals (P < 0.05) and maintained them at or above the peak levels achieved after peripheral insulin treatment. Central insulin infusion did not affect peripheral (plasma) insulin or glucose concentrations. LH pulse frequency in insulin-treated animals was greater than that in saline-treated animals (3.5 ± 0.2 vs. 2.3 ± 0.3 pulses/4 h, P < 0.01), but it was less than that during peripheral insulin treatment (4.8 ± 0.2 pulses/4 h, P < 0.01). Our findings suggest that physiologic levels of central insulin supplementation are able to increase pulsatile LH secretion in diabetic sheep with low peripheral insulin. These results are consistent with the notion that central insulin plays a role in regulating pulsatile GnRH secretion.

The regulation of ovarian steroidogenesis in vitro by coho salmon FSH and LH was investigated in intact coho salmon follicles and isolated follicular layers at various stages of oocyte maturation, from late vitellogenesis until the completion of germinal vesicle breakdown (GVBD). In granulosa layers from all stages, LH, but not FSH, stimulated 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) production. In theca-interstitial layers from all stages, FSH and LH stimulated steroid production, LH being more potent than FSH. The basal steroid output of intact follicles was significantly lower than that of isolated follicular layers, and their response to FSH and LH also differed. First, the intact follicles produced 17α-hydroxyprogesterone in response to FSH during the central germinal vesicle stage while theca-interstitial layers did not. Second, estradiol-17β production was not inhibited by LH during final oocyte maturation in intact follicles, as observed for granulosa layers. Our results indicate that LH is the determining factor regulating the production of the maturation-inducing steroid, 17,20β-P, and the induction of GVBD in the salmonid ovary. In summary, we have provided evidence for maturation-associated changes in the effects of FSH and LH in the salmonid ovary, which further supports the hypothesis that FSH and LH have distinct functions in the teleost ovary.

The proteasome engages in protein degradation as a regulatory process in biological transactions. Among other cellular processes, the proteasome participates in degradation of ubiquinated cyclins in mitosis. However, its role in meiosis has not been established. Resumption of meiosis in the oocyte involves the activation of maturation promoting factor (MPF), a complex of p34cdc2 and cyclin B. Inactivation of this factor, occurring between the two meiotic divisions, is associated with degradation of cyclin B. In this study, we examined the possible involvement of the proteasome in regulation of the exit from metaphase I in spontaneously maturing rat oocytes. We found that upon resumption of meiosis, proteasomes translocate to the spindle apparatus. We further demonstrated that specific inhibitors of proteasome catalytic activity, MG132 and lactacystin, blocked polar body extrusion. Chromosome and microtubule fluorescent staining verified that MG132-treated oocytes were arrested at metaphase I. Intervention of proteasomal action with this inhibitor also resulted in accumulation of cyclin B and elevated activity of MPF. These data demonstrate that proteasomal catalytic activity is absolutely essential for the decrease in MPF activity and completion of the first meiotic division. Its translocation to the spindle apparatus may facilitate the timely degradation of cyclin B.

A novel testis-specific protein, termed LRTP, was identified by screening both human and mouse testis and mouse pachytene spermatocyte cDNA libraries. Sequence analyses (GenBank accession number: AF092208) revealed that LRTP contains an amino terminus leucine-rich repeat domain. There are several acidic regions rich in glutamic acid in the C-terminus. The sequence, by GenBank search, shows similarities to LANP and SDS22 , leucine-rich repeat proteins localized to the nucleus and involved in the regulation of protein phosphatases. In mouse, the mRNA is first detected at about Day 14 postpartum, presumably when mid-pachytene spermatocytes are first seen. In situ hybridization confirmed the expression of the LRTP mRNA at this stage of spermatogenesis. Immunohistochemical analysis revealed that the protein is most abundant in the cytoplasm of pachytene and diplotene cells, corresponding to late prophase of meiosis I. Immunohistochemical localization is markedly reduced in secondary spermatocytes, suggesting a functional association of LRTP with meiosis. An LRTP cDNA probe did not bind to mouse ovary RNA in a dot blot assay.

The role of endometrial and embryonic integrins during implantation remains unresolved although work in animal models and in humans supports their involvement in this process. Temporal and spatial distribution of the α_vβ₃ integrin on both embryo and endometrium in women and mice coincides with the time of initial attachment during implantation. In mice, the endometrial and embryonic α_vβ₃ integrin is present at the time of implantation, as shown by reverse transcription-polymerase chain reaction and immunohistochemistry. In situ hybridization demonstrates the presence of the α_vβ₃ integrin on the subluminal stromal cells of the uterus. Functional blockade of this integrin on the day of implantation by intrauterine injection of neutralizing monoclonal antibodies against α_v or β₃ integrin subunits, arg-gly-asp (RGD)-containing peptides, or of the disintegrin echistatin, reduced the number of implantation sites compared to controls receiving BSA. These studies demonstrate that, like the human, the murine α_vβ₃ integrin is expressed at the time of implantation in the endometrium and on the blastocyst, and may play a critical role in the cascade of events leading to successful implantation.

In the present study we examined the involvement of interleukin (IL)-1α, -1β, FSH, and lipopolysaccharide (LPS) in the regulation of IL-1α and -1β production by Sertoli cells under in vitro conditions. Sertoli cell cultures from immature mice produced constitutively basal levels of intracellular IL-1α. Stimulation of Sertoli cell cultures with LPS (5 μg/ml) resulted in a maximal production of intracellular IL-1α 2 h after the stimulation. Thereafter, these levels decreased but remained significantly higher within 24 h after stimulation than those in control cultures. The effect of LPS on IL-1α production was dose dependent. FSH did not show any effect on intracellular IL-1α production by Sertoli cells. IL-1α could not be detected in supernatants of unstimulated or stimulated Sertoli cell cultures. Sertoli cell cultures stimulated with recombinant IL-1α induced optimal intracellular levels of IL-1α within 2 h of stimulation. These levels remained high 24 h after stimulation. However, stimulation of Sertoli cell cultures with IL-1β induced a peak of IL-1α production 8 h after stimulation. These levels decreased 24 h after the stimulation but were still found to be significantly higher than those in control cultures. The addition of IL-1 receptor antagonist (IL-1ra) to Sertoli cell cultures did not significantly alter their capacity to produce IL-1α. However, the stimulatory effects of recombinant IL-1α on IL-1α production by Sertoli cell cultures were reversed by the concomitant addition of recombinant IL-1ra.

No immunoreactive IL-1β could be detected in lysates or conditioned media of immature murine Sertoli cells under any of the stimulatory conditions outlined.

Our results may suggest the involvement of physiological (IL-1) and pathophysiological factors (LPS) in the regulation of spermatogenesis and spermiogenesis processes and male fertility.

We have investigated the effect of intrafetal cortisol administration, before the normal prepartum cortisol surge, on the expression of 11β hydroxysteroid dehydrogenase (11βHSD) type 2 mRNA in the fetal adrenal. We also determined whether increased fetal cortisol concentrations can stimulate growth of the fetal adrenal gland or increase expression of adrenal steroidogenic enzymes. Cortisol (hydrocortisone succinate: 2.0–3.0 mg in 4.4 ml/24 h) was infused into fetal sheep between 109 and 116 days of gestation (cortisol infused; n = 12), and saline was administered to control fetuses (saline infused; n = 13) at the same age. There was no effect of cortisol infusion on the fetal adrenal:body weight ratio (cortisol: 101.7 ± 5.3 mg/kg; saline: 108.2 ± 4.3 mg/kg). The ratio of adrenal 11βHSD-2 mRNA to 18S rRNA expression was significantly lower, however, in the cortisol-infused group (0.75 ± 0.02) compared with the group receiving saline (1.65 ± 0.14). There was no significant effect of intrafetal cortisol on the relative abundance of adrenal CYP11A1, CYP17, CYP21A1, and 3βHSD mRNA. A premature elevation in fetal cortisol therefore resulted in a suppression of adrenal 11βHSD-2. Increased intra-adrenal exposure to cortisol at this stage of gestation is, however, not sufficient to promote adrenal growth or steroidogenic enzyme gene expression.

We investigated the effect of retinoids on the development of Sertoli, germ, and Leydig cells using 3-day culture of testes from fetuses 14.5 and 18.5 days post-conception (dpc) and from neonates 3 days postpartum (dpp). Addition of 10⁻⁶ M and 3.10⁻⁸ M retinoic acid (RA) caused a dose-dependent disruption of the seminiferous cords in 14.5-day-old fetal testes, without any change in the 5-bromo-2′-deoxyuridine (BrdU) labeling index of the Sertoli cells. RA caused no disorganization of older testes, but it did cause hyperplasia of the Sertoli cells in 3-dpp testes. Fragmentation of the Sertoli cell DNA was not detected in control or RA-treated testes at any age studied. The cAMP produced in response to FSH was significantly decreased in RA-treated testes for all studied ages. Both 10⁻⁶ M and 3.10⁻⁸ M RA dramatically reduced the number of gonocytes per 14.5-dpc testis. This resulted from a high increase in apoptosis, which greatly exceeded the slight increase of mitosis. RA caused no change in the number of gonocytes in testes explanted on 18.5 dpc (the quiescent period), whereas it increased this number in testes explanted on 3 dpp (i.e., when gonocyte mitosis and apoptosis resume). Lastly, RA and retinol (RE) reduced both basal and acute LH-stimulated testosterone secretion by 14.5-dpc testis explants, without change in the number of 3β-hydroxysteroid dehydrogenase-positive cells per testis. Retinoids had no effect on basal or LH-stimulated testosterone production by older testes. In conclusion, RE and RA are potential regulators of the development of the testis and act mainly negatively during fetal life and positively during the neonatal period on the parameters we have studied.

Osteopontin (OPN) is an acidic phosphorylated glycoprotein component of the extracellular matrix that binds to integrins at the cell surface to promote cell-cell attachment and cell spreading. This matrix constituent is a ligand that could potentially bind integrins on trophectoderm and endometrium to facilitate superficial implantation and placentation. OPN mRNA increases in the endometrial glandular epithelium (GE) of early-pregnant ewes, and OPN protein is secreted into the uterine lumen. Therefore, progesterone and/or interferon-tau (IFNτ) may regulate OPN expression in the uterine GE. Cyclic ewes were ovariectomized and fitted with intrauterine (i.u.) catheters on Day 5 and treated daily with steroids (i.m.) and protein (i.u.) as follows: 1) progesterone (P, Days 5–24) and control serum proteins (CX, Days 11–24); 2) P and ZK 136.317 (ZK; progesterone receptor [PR] antagonist, Days 11–24) and CX proteins; 3) P and recombinant ovine IFNτ (roIFNτ, Days 11–24); or 4) P and ZK and roIFNτ. All ewes were hysterectomized on Day 25. Progesterone induced the expression of endometrial OPN mRNA in the GE and increased secretion of a 45-kDa OPN protein from endometrial explants maintained in culture for 24 h. Administration of ZK ablated progesterone effects. Intrauterine infusion of roIFNτ did not affect OPN gene expression or secretion in any of the steroid treatments. Interestingly, OPN mRNA-positive GE cells lacked detectable PR expression, although PR were detected in the stroma. Results indicate that progesterone regulates OPN expression in GE through a complex mechanism that includes PR down-regulation, and we suggest the possible involvement of a progesterone-induced stromal cell-derived growth factor(s) that acts as a progestamedin.

Culture of preantral follicles has important biotechnological implications through its potential to produce large quantities of oocytes for embryo production and transfer. A long-term culture system for bovine preantral follicles is described. Bovine preantral follicles (166 ± 2.15 μm), surrounded by theca cells, were isolated from ovarian cortical slices. Follicles were cultured under conditions known to maintain granulosa cell viability in vitro. The effects of epidermal growth factor (EGF), insulin-like growth factor (IGF)-I, FSH, and coculture with bovine granulosa cells on preantral follicle growth were analyzed. Follicle and oocyte diameter increased significantly (P < 0.05) with time in culture. FSH, IGF-I, and EGF stimulated (P < 0.05) follicle growth rate but had no effect on oocyte growth. Coculture with granulosa cells inhibited FSH/IGF-I-stimulated growth. Most follicles maintained their morphology throughout culture, with the presence of a thecal layer and basement membrane surrounding the granulosa cells. Antrum formation, confirmed by confocal microscopy, occurred between Days 10 and 28 of culture. The probability of follicles reaching antrum development was 0.19 for control follicles. The addition of growth factors or FSH increased (P < 0.05) the probability of antrum development to 0.55. Follicular growth appeared to be halted by slower growth of the basement membrane, as growing follicles occasionally burst the basement membrane, extruding their granulosa cells. In conclusion, a preantral follicle culture system in which follicle morphology can be maintained for up to 28 days has been developed. In this system, FSH, EGF, and IGF-I stimulated follicle growth and enhanced antrum formation. This culture system may provide a valuable approach for studying the regulation of early follicular development and for production of oocytes for nuclear/embryo transfer, but further work is required.

Previous studies demonstrated the presence of high-affinity GnRH binding sites and compounds with GnRH-like activity in the ovary of seabream, Sparus aurata, providing evidence for the role of GnRH as a paracrine/autocrine regulator of ovarian function in this species. In the present study, the expression of three forms of GnRH (salmon, chicken-II, and seabream) genes in this marine teleost species was demonstrated for the first time. Moreover, there is evidence for differential splicing and intronic expression of cGnRH-II and sbGnRH. Treatment of seabream follicle-enclosed oocytes with salmon GnRH stimulated reinitiation of oocyte meiosis, whereas chicken GnRH-II treatment was without effect. Novel information was also provided about organization of cGnRH-II and seabream GnRH transcripts, confirming that GnRH gene organization is maintained through evolution, despite changes in the size and sequence of exons and introns.

The objective of the present study was to determine the changes in follicular fluid steroid concentrations and in granulosa cell steroidogenic enzyme expression during the follicular phase, in relation to follicular size and physiological status in the mare.

Follicular fluid and follicular cells were recovered by ultrasound-guided follicular punctures either around the time of emergence of the dominant follicle, at the end of the dominant follicle growth, or at the preovulatory stage, after injection of gonadotropin to induce ovulation. Cellular relative amounts of steroidogenic acute regulatory protein (StAR), P450-side chain cleavage (P450_scc), 3β-hydroxysteroid dehydrogenase (3βHSD), 17α-hydroxylase, and aromatase were assessed by semiquantitative Western blot and densitometry. Follicular fluid was assayed for cholesterol concentrations by colorimetric assay and for progesterone, testosterone, and estradiol-17β concentrations by RIA.

Intrafollicular concentrations of progesterone and estradiol-17β significantly increased in the dominant follicle during growth. After injection of gonadotropin, follicular maturation was characterized by a decrease in estradiol-17β concentrations and a further increase in progesterone concentrations. Granulosa cells from dominant follicles had increased levels of StAR, P450_scc, 3βHSD, and aromatase during growth, but decreased levels during maturation. Levels of StAR, P450_scc, 3βHSD, and aromatase, as well as progesterone and estradiol-17β, were lower in granulosa cells from subordinate than from dominant follicles. We did not observe a relationship between the steroidogenic activity of follicles and the capacity of their enclosed oocytes to complete meiosis in vitro.

The cAMP-responsive element modulator (CREM) gene encodes a transcription factor that is essential for spermatogenesis. In mouse testis, several CREM repressors and activators have been identified. In contrast to the situation for the mouse, however, little is known about CREM isoforms in the primate testis. We analyzed CREM isoforms and mRNA expression in a clinically relevant primate model, the cynomolgus monkey (Macaca fascicularis). A cDNA library was generated from monkey testis; and two activator isoforms (τ2 with and without exon γ) were identified, which displayed high sequence identity to mouse and human isoforms. The insertion of exon γ was observed for the first time in the primate testis. CREM activator expression was confined to the testis, where it was seen in late pachytene spermatocytes and round spermatids in specific spermatogenic stages, as revealed by in situ hybridization. Comparison of the mRNA and the recently described protein expression indicated a lack of translational delay of CREM expression. Comparative analysis of testicular CREM expression by reverse transcription-polymerase chain reaction yielded several transcripts in the rat, mouse, hamster, and marmoset; two transcripts in cynomolgus and rhesus monkeys; and one transcript in men. These findings suggest an evolutionary trend from multiple activator isoforms to a single activator transcript in men.

The placenta secretes various factors in stage- and cell-specific manners. We have identified a cDNA encoding a novel protein with 124 amino acids, which was named spongiotrophoblast specific protein (SSP). SSP is highly homologous to mouse 4311, showing 81% and 59% similarity at the nucleotide and amino acid levels, respectively. Northern blot analysis showed that SSP mRNA was first detected on Day 14 of pregnancy, peaked on Day 16, and remained elevated until term. In situ hybridization analysis showed that SSP mRNA was specifically expressed in spongiotrophoblast cells of Day 20 placenta but not in Day 12 placenta. No expression was detected from the differentiated or undifferentiated rat choriocarcinoma Rcho-1 cell line. Native SSP was detected as a 19-kDa molecule by Western blotting in cell extracts prepared from the junctional zone. SSP was predicted to be a secretory protein, because 1) a hydropathy test revealed that SSP contained an N-terminal hydrophobic region and 2) native SSP was also detected in the cultured media of junctional zone explants. To further investigate a potential signal peptide of this protein, sets of recombinant SSP were generated using a COS7 transfection system. The N-terminal amino acid sequence of secreted recombinant SSP confirmed that the N-terminal 17 amino acids had been cleaved to produce a secretory protein. Thus, we have identified and cloned a novel secretory protein, SSP, which is specifically expressed by rat spongiotrophoblast cells during the latter half of pregnancy.

We have isolated the full-length coding sequence for mouse KIFC5A (kinesin family c-terminal 5A) cDNA, encoding a motor protein found in the testes. The complete sequence of the KIFC5A cDNA is homologous to a group of carboxyl-terminal motors, including hamster CHO2, human HSET, and mouse KIFC1 and KIFC4. The KIFC5A and KIFC1 cDNAs are nearly identical except for the presence of two additional sequence blocks in the 5′-end of KIFC5A and a number of single base-pair differences in their motor domains. Polymerase chain reaction amplification and sequencing of the 5′-end of KIFC5A identified 3 distinct RNA species in testes and other tissues. Sequence comparison and genetic mapping confirmed the existence of a small multi-gene family in the mouse and suggest possible mechanisms of alternative splicing, genetic duplication, and separate genetic loci in the generation of these motors. In order to examine the possible role of these motors in germ cells of the testes, an antibody to a shared epitope was used to localize this group of proteins to different spermatogenic cell types. These experiments suggest that KIFC5-like motor proteins are associated with multiple microtubule complexes in male germ cells, including the meiotic spindle, the manchette, and the flagella.

Intracellular calcium ([Ca² ]_i) rises are a hallmark of mammalian fertilization and are associated with normal activation of embryonic development. Injection of mammalian sperm cytosolic factor (SCF) into oocytes has been shown to trigger [Ca² ]_i rises similar to those observed during fertilization, and to initiate normal embryonic development. However, Ca² release has also been shown to be associated with cell death, but the mechanisms of the detrimental effects of Ca² stimulation on development have not yet been investigated. Thus, studies were undertaken using SCF to test the effects of [Ca² ]_i oscillations on oocyte activation in freshly ovulated and aged oocytes. Injections of 1 mg/ml SCF into freshly ovulated mouse metaphase II oocytes, which evoked Ca² responses with low frequency and short duration, induced normal activation and cleavage to the two-cell stage. Conversely, injection of 15 mg/ml SCF, which triggered high-frequency and persistent Ca² responses, induced abnormal activation that was characterized by abnormal chromatin configurations, inhibition of DNA synthesis, and lack of first mitotic spindle assembly. More importantly, fertilization-like Ca² responses induced by injection of 1 mg/ml SCF triggered cell death, rather than activation, in in vitro-aged oocytes. These oocytes exhibited extensive cytoplasmic and DNA fragmentation that was accompanied by activation of protein caspases, all of which are signs of apoptotic cell death. Fewer similarly aged oocytes that were either unstimulated or activated with 7% ethanol underwent fragmentation. Together, these results suggest that [Ca² ]_i oscillations are required to activate freshly ovulated oocytes, but if initiated at abnormally high frequency and duration or if induced in aged oocytes, the [Ca² ]_i oscillations may trigger premature termination of embryonic development.

In human spermatozoa, progesterone (P₄) induces a depolarization of the plasma membrane, a rapid calcium (Ca² ) influx, and a chloride efflux. The sodium ion (Na ) was partly responsible for the P₄-induced depolarizing effect but was not required for calcium influx. We used fluorescent probes for spectrofluorometry to investigate whether P₄ induced a Na influx and whether voltage-operated channels were involved in Na and/or Ca² entries. We found that 10 μM P₄ significantly increased intracellular Na concentration from 17.8 ± 2.0 mM to 27.2 ± 1.6 mM (P < 0.001). Prior incubation of spermatozoa with 10 μM flunarizine, a Na and Ca² voltage-dependent channel blocker, inhibited the sodium influx induced by 10 μM P₄ by 84.6 ± 15.4%. The Ca² influx induced by 10 μM P₄ was also significantly inhibited in a Na -containing medium by 10 μM flunarizine or 10 μM pimozide (P < 0.01). In contrast, flunarizine had no inhibitory effect on the Ca² influx induced by 10 μM P₄ in spermatozoa incubated in Na -depleted medium. The P₄-promoted acrosome reaction (AR) was significantly higher when spermatozoa were incubated in Na -containing medium as compared to Na -depleted medium. These data demonstrate that P₄ stimulates a Na influx that could be involved in the AR completion. They also suggest that voltage-dependent Na and Ca² channels are implicated in P₄-mediated signaling pathway in human spermatozoa.

The effects of estrogen (E₂), progesterone (P₄), and E₂ and P₄ (E₂ P₄) on uterine, vaginal, and cerebellar nitric oxide synthase (NOS) were examined. Additionally, experiments were done to investigate whether NOS-containing nerves were present in the uterus and vagina and the extent to which vaginal smooth muscle response was dependent on nitric oxide (NO). Cytosolic NOS was determined by the formation of [¹⁴C]citrulline from [¹⁴C]arginine, and NOS localization was visualized by immunohistochemistry. Vaginal smooth muscle relaxation was induced by electrical field stimulations (EFS). NOS activity in the uterus was markedly down-regulated in all hormone-treated groups. Vaginal NOS activity was nearly 4-fold higher than the uterine NOS activity and was considerably reduced by E₂ or E₂ P₄ treatment. In contrast to findings in the uterus, P₄ treatment up-regulated vaginal NOS. Hormone treatment had no significant effect on cerebellar NOS. NOS-containing nerves could be demonstrated in the uterus and vagina by immunohistochemistry. Vaginal smooth muscle responded with relaxation after EFS, which was inhibited by N^G-nitro-l-arginine. A relatively high vaginal NOS, a down-regulation by E₂, an up-regulation by P₄, and NO-dependent response of vaginal smooth muscle suggest a tissue-specific physiological role.

The cyclin-dependent kinase (CDK) inhibitor p27, the regulator of the cell cycle, is required for proper functioning of luteinizing/luteinized cells in vivo. Since different members of the CDK family may be targeted by p27 during luteinization-associated cell cycle exit, this in vivo study further analyzed the organization of the network of cell cycle regulators that may underlie both the establishment and maintenance of the luteal phenotype. Most importantly, it shows that the luteinization process is associated with down-regulation of CDK2 and cyclin D1, and up-regulation of p27 and cyclin D3. Both p27 and cyclin D3 proteins not only accumulated during initial phases of luteinization, but they remained elevated until termination of the luteal function. Along with its accumulation, p27 lost physical contact with CDK2 and instead became associated with CDK4. In fully luteinized cells, all cyclin D3 was incorporated into complexes with p27, some complexes being p27/cyclin D3/CDK4 trimers. Despite the significant amounts of CDK4 and CDK6, only nonphosphorylated forms of retinoblastoma protein were detectable in fully luteinized cells. Together, our data indicate that while inhibition of proliferation is underlaid by the progressive loss of positive regulators of the cell cycle, including cyclins and CDK2, maintenance of the luteal phenotype is driven by up-regulated levels of p27 and cyclin D3, at least partially owing to formation of p27/cyclin D3/CDK4 trimers.

Horse oocytes were collected from an abattoir over a 15-mo period. After classification of follicle size and cumulus morphology, oocytes were either fixed immediately (0 h) or matured in vitro (24 h). There was no effect of season on the number of antral follicles present on the ovaries, or on oocyte maturation rate for any class of oocyte. The proportion of oocytes having condensed chromatin at 0 h increased with increasing follicle size. The oocyte maturation rate also increased with follicle size, and for follicles ≤ 20-mm diameter, was higher for oocytes initially having expanded cumuli than for those having compact cumuli. The maturation rate was strongly correlated (r² = 0.92) with the proportion of oocytes having condensed chromatin at 0 h. Oocytes with diffuse chromatin were found essentially only in follicles ≤ 20-mm diameter that yielded compact granulosa, indicating follicle viability. Presence of diffuse chromatin was inversely related to maturation rate. We conclude that the major signal for chromatin condensation, and thus acquisition of meiotic competence, occurs in viable follicles after 20-mm diameter in the horse. Condensation of chromatin in oocytes in smaller apparently viable follicles, while associated with acquisition of meiotic competence, may represent a pre-atretic change.

Two experiments were conducted to determine whether the failure of males to induce sexual activity in goats during seasonal anestrus is due to unresponsiveness of females to male stimulus or insufficient stimulation from males. In the first study, one group of males (sexually inactive, SI; n = 4) was kept under natural photoperiod while the other (sexually active, SA; n = 4) was subjected to 2.5 mo of long days (16L:8D) and received 2 s.c. implants of melatonin. Two mo later, 2 different flocks of anovulatory goats previously separated from bucks were exposed to either SI (n = 34) or SA (n = 40) bucks. Progesterone assays and estrous behavior were used to determine ovarian and behavioral responses of the females to teasing. Of the goats exposed to SI males, only 2 ovulated, and none showed estrous behavior during the 35 days of the study. In contrast, all females (40 of 40) in contact with SA males ovulated and showed at least one estrous behavior during the first 11 days following male introduction (P < 0.001). Overall, 38 of 40 females stimulated with SA bucks were diagnosed pregnant at Day 35, according to progesterone assay (versus 0 in SI-treated group: P < 0.001). To control for a possible difference of responsiveness between flocks, the experiment was repeated 1 yr later using a single flock of goats divided into 2 groups. Again, over the first 14 days, 1 of 33 goats showed estrous behavior in the SI-treated group versus 27 of 33 in the SA-treated group (P < 0.001). Therefore, treating bucks with long days and melatonin increased their teasing capacity to induce sexual activity in females during anestrus. These results indicate that the absence of response to teasing at this time of the year is not due to female unresponsiveness, but to insufficient stimulation from the male.

The insulin-like growth factor (IGF) system plays an important role in the regulation of uterine function and placental growth. However, there is little information regarding the localization and regulation of IGF binding protein-5 (IGFBP-5) in the reproductive tract. The distribution of this IGFBP was therefore investigated using in situ hybridization in sections of utero-placental tissue obtained throughout the estrous cycle, up to Day 55 of gestation, and on Days 16–17 from both horns of ewes with unilateral pregnancies that followed uterine transection. In nonpregnant ewes, IGFBP-5 mRNA was present at high concentrations in the maternal caruncles and luminal epithelium, and at moderate levels in myometrium. In these regions IGFBP-5 mRNA showed cyclic variations, with concentrations peaking around ovulation, whereas low expression in the endometrial stroma remained constant. During pregnancy, there was additional localization to the endometrial glands; and in all regions, with the exception of the caruncles, concentrations increased significantly with gestational age. In transected uteri, concentrations in the luminal epithelium of the pregnant horn were significantly higher than those in the nonpregnant horn. In the caruncles, IGFBP-5 mRNA formed an intense band just below the tips of the invading fetal villi. Below this band, IGFBP-5 mRNA localized to form a series of rings, which could create a route to allow the fetal villi access into the caruncular stroma for nutrient exchange. In conclusion, IGFBP-5 is abundantly expressed in the ovine reproductive tract, with both the concentration and localization differentially regulated during the cycle and pregnancy.

To determine whether gestation-related changes in responsiveness of the rat uterus to β-adrenergic agonists are mediated at the level of adenylyl cyclase, we measured myometrial adenylyl cyclase activity and protein quantities during pregnancy and labor. In rat myometrial membranes, basal adenylyl cyclase activity increased from the nonpregnant state to mid (Days 12–14) and then late (Days 18–20) gestation and then decreased intrapartum (Day 22). Stimulated adenylyl cyclase activity, at the level of the β-adrenergic receptor (isoproterenol, 10⁻⁴ M), the G protein (GTP, 10⁻⁵ M), or the adenylyl cyclase enzyme (MnCl₂, 20 mM), was similarly altered during gestation. Total adenylyl cyclase protein was quantified by [³H]forskolin binding assay in myometrial membranes from nonpregnant and pregnant (Day 14, Day 20, Day 21, and intrapartum Day 22) rats. Adenylyl cyclase protein increased progressively from nonpregnant rats to pregnant rats at mid (Day 14) and late (Day 20) gestation, but it decreased abruptly to nonpregnant levels on Day 21, the day before parturition, and remained at similar levels on Day 22 (intrapartum). The gestation-related increase in expression of myometrial adenylyl cyclase protein may facilitate uterine quiescence during pregnancy, and the abrupt decrease of adenylyl cyclase protein on the last day of pregnancy may be a contributing mechanism for the initiation of labor.

Testicular inflammation is classically observed in pathogenesis caused by infectious agents, environmental toxins, trauma, or autoimmune reactions and can lead to transitory or even permanent sterility. In these situations, a leukocyte infiltration is generally encountered. Macrophage inflammatory proteins (MIP)-1α and −1β and monocyte chemoattractant protein-1 (MCP-1) are CC-chemokines involved in macrophage and lymphocyte chemoattraction. In the present study, using reverse transcription-polymerase chain reaction, Northern blot, and a specific ELISA, we investigated whether or not these chemokines are present within the testis and whether they are induced by a number of proinflammatory cytokines and lipopolysaccharides (LPS). MIP-1α and MIP-1β were not detected in Sertoli cells, germ cells, peritubular cells, or Leydig cells. In contrast, MCP-1 mRNA and protein were found to be expressed by control isolated peritubular cells, and expression was markedly stimulated by interleukin-1α and−1β (IL-1α and IL-1β), tumor necrosis factor α (TNF-α), interferon γ, and LPS. Leydig cells expressed MCP-1 when stimulated by IL-1β. In contrast, MCP-1 was not found to be produced by Sertoli cells or germ cells as established by Northern blot and ELISA techniques. The kinetics of MCP-1 production by peritubular cells, as demonstrated by expression as early as 8 h poststimulation, are compatible with there being a rapid mobilization of these cells and this chemokine in an inflammatory process. Moreover, MCP-1 production by peritubular cells after half-maximal stimulation by LPS, TNF-α, and IL-1β (2 pg/ml–0.9 ng/ml) is also compatible with the physiologic concentrations of the proinflammatory cytokines generally found in an inflammatory site. It is concluded that MCP-1 is produced by Leydig cells and peritubular cells and that it could be involved in the mobilization and migration of leukocytes observed during testicular inflammation.

Nutritionally induced anovulatory and cyclic Angus × Hereford heifers were used to evaluate follicular growth and concentrations of hormones and metabolites during anovulation and resumption of ovulation. Anovulatory heifers were fed to gain 0.6 (LGAIN) or 1.5 (HGAIN) kg/day until resumption of ovulation, and heifers with normal estrous cycles were fed a maintenance diet (M). Follicles ≥ 4 mm in diameter were measured by daily ultrasonography in HGAIN and LGAIN heifers during one follicular wave before realimentation (Wan) and in two waves (W-2, W-1) immediately before the wave resulting in first ovulation or luteinization (W0). Ovaries of M heifers were evaluated to determine the day of ovulation of the second-wave dominant follicle (DF). Resumption of ovulation after realimentation occurred 23 days earlier in HGAIN than in LGAIN. Maximum diameter, growth rate, and persistence of dominant follicles increased, while persistence of first subordinate follicles decreased between anovulation and resumption of ovulation in anovulatory heifers. Concentrations of LH in serum were similar for HGAIN and LGAIN and gradually increased during realimentation. The increase in estradiol before the first ovulation was less in realimented heifers compared with cyclic heifers. Concentrations of insulin-like growth factor-I (IGF-I) in HGAIN and LGAIN gradually increased during realimentation but were lower than concentrations of IGF-I in cyclic heifers at ovulation. Increased diameter, growth rate, and persistence of the DF were associated with increased concentrations of LH, estradiol, and IGF-I during the transition from nutritionally induced anovulation to resumption of ovulatory cycles.

Maternal administration of androstenedione produces a sustained fall in maternal plasma adrenocorticotropic hormone (ACTH) concentrations in the pregnant nonhuman primate. We hypothesize a negative feedback influence on the maternal hypothalamo-pituitary-adrenal (HPA) axis by androgens in primates. This may reflect an important maternal adaptation during pregnancy in primates preventing premature induction of labor by maternal stress. However, androstenedione is precursor for placental estradiol-17β synthesis, and infusion of androstenedione into pregnant primates elevates maternal plasma estradiol-17β to term concentrations. Thus, it could be argued that 1) the effects attributed to androstenedione on the maternal HPA axis are mediated by estrogen rather than by androgen and 2) the negative influence of androgens may be on placental ACTH rather than, or in addition to, pituitary ACTH. To discriminate between androgenic and estrogenic effects of androstenedione on pituitary and/or placental ACTH function in primates we measured plasma ACTH, cortisol, and dehydroepiandrosterone sulfate (DHEAS) concentrations in nonpregnant baboons after treatment with either androstenedione or estradiol-17β.

Nine female baboons were studied between 14 and 22 days postpartum prior to estrous cycling. After 2 days of baseline, a continuous i.v. infusion of androstenedione (1.5 mg/kg per h in 10% intralipid, IL) was started at 0900 h and maintained for 9 days in 3 baboons. A similar protocol was carried out in another 3 baboons that received a continuous i.v. infusion of estradiol-17β (10 μg/kg per h in 10% IL) instead of androstenedione. Three additional baboons received continuous i.v. IL vehicle alone and served as controls. Arterial blood samples (0.5 ml) for measurement of plasma hormones were taken during baseline and after 1, 3, 5, 7, and 9 days of infusion.

Baseline plasma ACTH, DHEAS, and cortisol concentrations were similar among all groups. Plasma ACTH did not change during IL, increased following estradiol-17β, and fell during androstenedione treatment. Accordingly, plasma cortisol and DHEAS concentrations were also unaltered by IL, and both steroids increased during estradiol-17β treatment. In contrast, plasma cortisol and DHEAS remained unaltered from baseline during androstenedione treatment, despite the fall in plasma ACTH measured at this time.

These data in the nonpregnant baboon 1) are consistent with negative feedback on pituitary ACTH by androgens and 2) demonstrate a positive influence on pituitary-adrenal function by estrogen in primates.

Simultaneous measurements of plasma LH, body temperature, and locomotor activity were made in laying turkey hens and are reported. Blood samples were remotely collected using a jugular cannula system, and body temperature and locomotor activity were remotely monitored using a radiotelemetry system in freely moving laying turkeys. Under a photoschedule of 14L:10D, the period for preovulatory surges of LH was 25.7 ± 0.4 h while the periods for peak body temperature and onset of sustained locomotor activity were 24.9 ± 0.4 and 25.7 ± 0.5 h, respectively. During exposure to constant light, the periods for preovulatory surges of LH, peak body temperature, and onset of sustained locomotor activity increased to 27.9 ± 0.9, 26.7 ± 0.7, and 27.4 ± 0.7 h, respectively. With the 14L:10D photoschedule, initiation of LH surges was restricted to the scotophase, but after 8 days of constant light, initiation of LH surges had dispersed throughout the 24-h subjective day and night. With constant light, the amplitude of the peak body temperature rhythm decreased, while the duration of the locomotor activity rhythm became broadened and, in some birds, disorganized. Peak body temperature and onset of locomotor activity rhythms and LH surges did not coincide, even though peak body temperature, onset of locomotor activity, and LH surges had similar periods. It is concluded that 1) the photoschedule influences the periods of the LH surge, peak body temperature, and onset of locomotor activity; and 2) a specific or direct relationship between the rhythms of LH surge, body temperature, and locomotor activity remains to be determined in laying turkey hens.

The objective of this study was to compare the ultrastructure of bovine compact morulae produced in vivo or in vitro using morphometric analysis. Compact morulae produced in vivo were obtained from superovulated Holstein cows. Compact morulae produced in vitro were obtained from cumulus-oocyte complexes aspirated from ovaries of Holstein cows. The complexes were matured and fertilized in vitro. At 20 h postinsemination (hpi), zygotes were distributed into 1 of 3 culture media: 1) IVPS (in vitro produced with serum): TCM-199 10% estrous cow serum (ECS); 2) IVPSR (in vitro produced with serum restriction): TCM-199 1% BSA until 72 hpi followed by TCM-199 10% ECS from 72 to 144 hpi; 3) mSOF (modified synthetic oviductal fluid): SOF 0.6% BSA. At 144 hpi, five grade 1 compact morulae from each of the four treatments were prepared for transmission electron microscopy. The volume density occupied by cellular components was determined by the point-count method using a sampling of seven to nine random micrographs from each compact morula. The volume density of lipid was greater (P < 0.05) in compact morulae from IVPS, IVPSR, and mSOF treatments compared with those produced in vivo. There was a reduced proportional volume of total mitochondria in compact morulae from the IVPS treatment compared with those produced in vivo (P < 0.05). For compact morulae from the IVPS culture treatment, the volume density of vacuoles was greater than that for compact morulae produced in vivo (P < 0.05). The cytoplasmic-to-nuclear ratio for compact morulae from the IVPS treatment was increased (P < 0.05) compared with the ratio for those produced in vivo. In conclusion, compact morulae produced in vitro differed ultrastructurally from those produced in vivo. Compact morulae produced in IVPS culture medium possessed the greatest deviations in cellular ultrastructure.