Lysophosphatidic acid (LPA) belongs to a new family of lipid mediators that are endogenous growth factors and that elicit diverse biological effects, usually via the activation of G protein-coupled receptors. LPA can be generated after cell activation through the hydrolysis of preexisting phospholipids in the membranes of stimulated cells. A dramatic elevation of LPA levels was found in serum of patients suffering from ovarian carcinoma. Because these high LPA amounts can be detected as early as stage I of the disease, LPA has been introduced as a new marker for ovarian cancer. Progression of the malignancy is correlated with a differential expression of various LPA receptor subtypes. The presence of LPA in the follicular fluid of healthy individuals implicates that this biological mediator may be relevant to normal ovarian physiology. LPA induces proliferation and mitogenic signaling of prostate cancer cells, and a novel LPA receptor isoform has been recognized in healthy prostate tissues. This evidence indicates multiple roles for LPA in both male and female reproductive physiology and pathology. In this review, we summarize the literature on LPA generation, the way it is degraded, and the mechanisms by which signals are transduced by various LPA receptors in reproductive tissues, and we discuss possible future research directions in these areas.

The zona pellucida (ZP) is an extracellular coat synthesized and secreted by the oocyte during follicular development and surrounding the plasma membrane of mammalian eggs. To date, the mechanism of synthesis and secretion, mode of assembly, and intracellular trafficking of the ZP glycoproteins have not been fully elucidated. Using antibodies against mouse ZP1, ZP2, and ZP3 in conjunction with the protein A-gold technique, we have shown an association of immunolabeling with the Golgi apparatus, secretory granules, and a complex structure called vesicular aggregate, respectively, in mouse ovarian follicles. In contrast, the neighboring granulosa cells were not reactive to any of the three antibodies used. Immunolabeling of ZP1, ZP2, and ZP3 was detected throughout the entire thickness of the ZP, irrespective of the developmental stage of ovarian follicles. Double and triple immunolocalization studies, using antibodies tagged directly to different sizes of gold particles, revealed an asymmetric spatial distribution of the three ZP glycoproteins in the zona matrix at various stages of follicular development. All three glycoproteins were specifically localized over small patches of darkly stained flocculent substance dispersed throughout the zona matrix. Very often, ZP1, ZP2, and ZP3 were found in close association. These results confirm findings from previous studies demonstrating that ovarian oocytes and not granulosa cells are the only source for mouse ZP glycoproteins. In addition, results from our morphological and immunocytochemical experiments suggest that the vesicular aggregates in the ooplasm are likely to serve as an intermediary in the synthesis and secretion of ZP glycoproteins. The stoichiometric disposition of ZP1, ZP2, and ZP3 in the zona matrix as revealed by double and triple immunolocalization studies provide further insight into some of the unanswered questions pertinent to the current model of mouse ZP structure proposed by the Wassarman group.

Transgenic mice carrying rat androgen-binding protein (ABP) genomic DNA express high amounts of testicular ABP and develop a progressive impairment of spermatogenesis. To understand the mechanism of these changes, we have studied the pattern of testicular germ cell proliferation from 7 to 360 days of age in wild-type (WT) control and transgenic homozygous (ABP-TG) mice by flow cytometry after labeling DNA in isolated germ cells with propidium iodide. At all ages studied, the body weight of the ABP-TG mice was lower than that of age-matched WT controls. Significantly reduced testicular weight and total germ cell number in the ABP-TG mice were evident from Day 30 and Day 60, respectively. Flow cytometric analysis of isolated germ cells revealed that the number of germ cells undergoing proliferation (S-phase cells) was identical in WT control and ABP-TG mice up to Day 14. Subsequently, the number of germ cells in S-phase was consistently higher in ABP-TG than in WT mice. The number of primary spermatocytes was significantly increased starting from Day 60, and the numbers of round and elongated spermatids were significantly reduced in the ABP-TG animals from Day 21 and Day 60 onwards, respectively. Immunocytometry for intracellular ABP at 90 days of age revealed that the percentage of ABP-containing germ cells was greater in ABP-TG than in WT mice. The continuous presence of ABP in mouse seminiferous tubules at greater than physiological concentrations facilitates the formation of primary spermatocytes but impairs subsequent transformation to round and elongated spermatids. Based on our observations and the analysis of the available literature, the most likely mechanism for production of these effects is sustained reduction in the bioavailability of androgens.

Host cell infection by sexually transmitted disease (STD)-causing microbes and fertilization by spermatozoa may have some mechanisms in common. If so, certain noncytotoxic agents could inhibit the functional activity of both organisms. High molecular mass poly(sodium 4-styrenesulfonate) (T-PSS) may be one of these compounds. T-PSS alone (1 mg/ml) or in a gel (2% or 5% T-PSS) completely prevented conception in the rabbit. Contraception was not due to sperm cytotoxicity or to an effect on sperm migration. However, T-PSS inhibited sperm hyaluronidase (IC₅₀ = 5.3 μg/ml) and acrosin (IC₅₀ = 0.3 μg/ml) and caused the loss of acrosomes from spermatozoa (85% maximal loss by 0.5 μg/ml). T-PSS (5% in gel) also reduced sperm penetration into bovine cervical mucus (73% inhibition by 1 mg gel/ml). T-PSS (5% in gel) inhibited human immunodeficiency virus (HIV; IC₅₀= 16 μg gel/ml) and herpes simplex viruses (HSV-1 and HSV-2; IC₅₀ = 1.3 and 1.0 μg gel/ml, respectively). The drug showed high efficacy against a number of clinical isolates and laboratory strains. T-PSS (5% in gel) also inhibited Neisseria gonorrhea (IC₅₀ < 1.0 gel/ml) and Chlamydia trachomatis (IC₅₀ = 1.2 μg gel/ml) but had no effect on lactobacilli. These results imply that T-PSS is an effective functional inhibitor of both spermatozoa and certain STD-causing microbes. The noncytotoxic nature should make T-PSS safe for vaginal use. T-PSS was nonmutagenic in vitro and possessed an acute oral toxicity of >5 g/kg (rat). Gel with 10% T-PSS did not irritate the skin or penile mucosa (rabbit) and caused no dermal sensitization (guinea pig). Vaginal administration of the 5% T-PSS gel to the rabbit for 14 consecutive days caused no systemic toxicity and only mild (acceptable) vaginal irritation. T-PSS in gel form is worthy of clinical evaluation as a vaginal contraceptive HIV/STD preventative.

Nuclear transfer to produce cattle is inefficient because 1) donor cells are not easily synchronized in the proper phase of the cell cycle, 2) the nucleus of these cells is not effectively reprogrammed, 3) the rate of attrition of late-term pregnancies is high, and 4) the health of early postnatal calves is compromised. The cyclin dependent kinase 2 inhibitor, roscovitine, was used to maximize cell cycle synchrony and to produce cells that responded more reliably to nuclear reprogramming. Roscovitine-treated adult bovine granulosa cells (82.4%) were more efficiently synchronized (P < 0.05) in the quiescent G0/G1 phase of the cell cycle than were serum-starved cells (76.7%). Although blastocyst development following nuclear transfer was elevated (P < 0.05) in the serum-starved group (21.1%) relative to the roscovitine-treated cells (11.8%), the number of cells in the blastocysts derived from roscovitine-treated cells was higher (P < 0.05) than those derived from the serum-starved group (roscovitine-treated group: 142.8 ± 6.0 cells; serum-starved group: 86.8 ± 14.5 cells). The resulting fetal and calf survival after embryo transfer was enhanced in the roscovitine-treated group (seven surviving calves from six pregnancies) compared with serum-starved controls (two calves born, one surviving beyond 60 days, from five pregnancies). Roscovitine culture can predictably synchronize the donor cell cycle and increase the nuclear reprogramming capacity of the cells, resulting in enhanced fetal and calf survival and increased cloning efficiency.

The evaluation of culture medium for bovine oocytes has progressed toward more defined conditions during the last few years. The main objective of this study was to evaluate different sources of albumin as a protein supplement during in vitro maturation (IVM) of bovine oocytes in synthetic oviduct fluid medium (SOF). The replacement of protein with polyvinyl pyrrolidone (PVP) or polyvinyl alcohol (PVA) was also evaluated. The effect of recombinant human FSH on cumulus expansion and nuclear maturation in SOF containing BSA (BSA-V) or PVP-40 was also studied. Addition of BSA-V during IVM retarded nuclear maturation when compared with addition of PVP-40 or use of SOF alone. The inclusion of different concentrations of BSA-V, fetal calf serum (FCS), or PVA during IVM had no positive effect on the developmental capacity of the oocytes compared with the use of SOF alone with no supplement but significantly decreased the percentage of embryos reaching the morula and blastocyst stages. However, when BSA-V was replaced with purified BSA, BSA that was essentially free of fatty acids, or chicken egg albumin, embryonic development rates were restored. The presence of PVP-40 but not PVP-360 during IVM significantly increased morula and blastocyst production. These results indicate that although SOF alone can support bovine oocyte maturation, a high proportion of morulae and blastocysts can be produced from IVM oocytes cultured in medium containing PVP-40. These studies are the first to show that the effect of FSH on nuclear maturation and cumulus expansion is dependent on substrates present in IVM medium.

Steroidogenesis is a major function of the developing follicle. However, little is known about the stage of onset of steroid regulatory proteins during follicular development in sheep. In this study, several steroidogenic enzymes were studied by immunohistochemistry and/or in situ hybridization; cytochrome P450 side chain cleavage (P450_scc), cytochrome P450 17α-hydroxylase (17αOH), 3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P450 aromatase (P450_arom), steroidogenic factor 1 (SF-1), steroidogenic acute regulatory protein (StAR), and LH receptor (LH-R). To define the stages of follicular growth, ovarian maps were drawn from serial sections of ovine ovaries, and follicles were located and classified at specific stages of growth based on morphological criteria. In this way, the precise onset of gene expression with respect to stages of follicular growth for all these proteins could be observed. The key findings were that ovine oocytes express StAR mRNA at all stages of follicular development and that granulosa cells in follicle types 1–3 express 3β-HSD and SF-1. Furthermore, the onset of expression in theca cells of StAR, P450_scc, 17αOH, 3β-HSD, and LH-R occurred in large type 4 follicles just before antrum formation. This finding suggests that although the theca interna forms from the type 2 stage, it does not become steroidogenically active until later in development. These studies also confirm that granulosa cells of large type 5 follicles express SF-1, StAR, P450_scc, LH-R, and P450_arom genes. These findings raise new questions regarding the roles of steroidogenic regulatory factors in early follicular development.

Although Smad 3 is known to serve as a signaling intermediate for the transforming growth factor beta (TGFβ) family in nonreproductive tissues, its role in the ovary is unknown. Thus, we used a recently generated Smad 3-deficient (Smad 3−/−) mouse model to test the hypothesis that Smad 3 alters female fertility and regulates the growth of ovarian follicles from the primordial stage to the antral stage. In addition, we tested whether Smad 3 affects the levels of proteins that control apoptosis, survival, and proliferation in the ovarian follicle. To test this hypothesis, breeding studies were conducted using Smad 3−/− and wild-type mice. In addition, ovaries were collected from Smad 3−/− and wild-type mice on Postnatal Days 2–90. One ovary from each animal was used to estimate the total number of primordial, primary, and antral follicles. The other ovary was used for immunohistochemical analysis of selected members of the B-cell lymphoma/leukemia-2 family of protooncogenes (Bax, Bcl-2, Bcl-x), proliferating cell nuclear antigen (PCNA), and cyclin-dependent kinase 2 (Cdk-2). The results indicate that Smad 3−/− mice have reduced fertility compared with wild type mice. The results also indicate that Smad 3 may not affect the size of the primordial follicle pool at birth, but it may regulate growth of primordial follicles to the antral stage. Further, the results indicate that Smad 3 may regulate the expression of Bax and Bcl-2, but not Bcl-x, Cdk-2, and PCNA. Collectively, these data suggest that Smad 3 may play an important role in the regulation of ovarian follicle growth and female fertility.

We studied the impact of prenatal androgen exposure on the timing of onset of puberty, maintenance of cyclicity in the first breeding season, and the LH surge mechanism in female sheep. Pregnant sheep were injected with testosterone propionate (100 mg i.m.) twice each week from Day 30 to Day 90 (D30–90) or from Day 60 to Day 90 (D60–90) of gestation (term = 147 days). Concentrations of plasma progesterone and gonadotropins were measured in blood samples collected twice each week from control (n = 10), D60–90 (n = 13), and D30–90 (n = 3) animals. Rate of weight gain and initiation of estrous behavior were also monitored. After the first breeding season, when the animals entered anestrus, competency of the gonadotropin surge system to respond to estradiol positive feedback was tested in the absence or presence of progesterone priming for 12 days. Prenatally androgenized females had similar body weight gain and achieved puberty (start of first progestogenic cycle) at the same time as controls. Duration of the breeding season and the number of cycles that occurred during the first breeding season were similar between control and prenatally androgenized sheep. In contrast, prenatal exposure to androgens compromised the positive feedback effects of estradiol. Onset of LH/FSH surges following the estradiol stimulus was delayed in both groups of androgenized ewes compared with the controls in both the absence and presence of progesterone priming. In addition, the magnitude of LH and FSH surges in the two animals that surged in the D30–90 group were only one third and one half, respectively, of the magnitudes observed in the control and D60–90 groups. The present findings indicate that disruption of the surge system can account for the fertility problems that occur during adulthood in prenatally androgenized sheep.

Relaxin-like factor (RLF) is a new member of the insulin-relaxin gene family known to be expressed in the ovarian follicular thecal cells of ruminants. To investigate the pattern of RLF expression in development and atresia of bovine follicles, antisera were raised in rats and rabbits to recombinantly expressed bovine pro-RLF and to chemically synthesized ovine RLF B chain, respectively. On dot blotting analysis, the rat anitserum bound to pro-RLF and less strongly to a synthetic mature ovine RLF lacking the C-domain, whereas the rabbit antiserum bound the mature form of ovine RLF. These antisera were used to immunostain bovine ovarian follicles of differing sizes and stages of health and atresia. 3β-Hydroxysteroid dehydrogenase was colocalized with pro-RLF (n = 86 follicles), and cholesterol side-chain cleavage cytochrome P450 was localized in another section of many of the same follicles (n = 66). Not all follicles expressed pro-RLF in the theca interna, so the results are presented as the proportion of follicles expressing pro-RLF. Both mature and pro-RLF were immunolocalized to steroidogenic thecal cells of healthy follicles. As follicles enlarged to >5 mm, the proportion expressing pro-RLF declined (19/19 for <5 mm and 18/26 for >6 mm). Atresia was divided into antral (antral granulosa cells dying first) or basal (basal cells dying first) and further divided into early, middle, and late. For antral atresia of small follicles (2–5 mm), no decline in the proportion expressing pro-RLF was observed (early 6/6, middle 2/2) until the late stages (1/4). For basal atresia, which only occurs in small follicles (2–5 mm), the proportion expressing pro-RLF declined in the middle (2/5) and late (0/8) stages. In larger follicles (>6 to <10 mm), the proportion expressing pro-RLF also declined with atresia (1/13). These declines in RLF expression with atresia or increasing size were not accompanied by a decline in the expression of steroidogenic enzymes in the theca interna. A significant (P < 0.001) inverse relationship in the expression of pro-RLF and 3β-hydroxysteroid dehydrogenase in the membrana granulosa was observed. We conclude that the expression of pro-RLF in the theca interna is switched off as follicles enlarge or enter atresia, whereas the expression of steroidogenic enzymes is maintained in the theca interna.

Adult stem cells maintain several self-renewing systems and processes in the body, including the epidermis, hematopoiesis, intestinal epithelium, and spermatogenesis. However, studies on adult stem cells are hampered by their low numbers, lack of information about morphologic or biochemical characteristics, and absence of functional assays, except for hematopoietic and spermatogonial stem cells. We took advantage of the recently developed spermatogonial transplantation technique to analyze germ line stem cells of the rat testis. The results indicate that the stem cell concentration in rat testes is 9.5-fold higher than that in mouse testes, and spermatogenic colonies derived from rat donor testis cells are 2.75 times larger than mouse-derived colonies by 3 mo after transplantation. Therefore, the extent of spermatogenesis from rat stem cells was 26-fold greater than that from mouse stem cells at the time of recipient testis analysis. Attempts to enrich spermatogonial stem cells in rat testis populations using the experimental cryptorchid procedure were not successful, but selection by attachment to laminin-coated plates resulted in 8.5-fold enrichment. Spermatogonial stem cells are unique among adult stem cells because they pass genetic information to the next generation. The high concentration of stem cells in the rat testis and the rapid expansion of spermatogenesis after transplantation will facilitate studies on stem cell biology and the introduction of genetic modifications into the male germ line. The functional differences between spermatogonial stem cells of rat vs. mouse origin after transplantation suggest that the potential of these cells may vary greatly among species.

Bax is a multidomain, proapoptotic member of the Bcl-2 family that is required for normal spermatogenesis in mice. Despite its proapoptotic function, previous results found that Bax-deficient mature male mice demonstrate increased cell death and dramatic testicular atrophy. The present study examined the role of Bax during the normal development of the testis to determine whether the increased cell death in mature mice could be explained by decreased apoptosis earlier in development. Consistent with this hypothesis, testicular atrophy is preceded by increased testicular weight and hypercellular tubules in immature Bax-deficient mice. TUNEL staining at Postnatal Day (P) 7 and morphological quantitation between P5 and P15 demonstrates decreased germ cell apoptosis in Bax-deficient mice. By P15, increased numbers of type A spermatogonia, and at P12 and P15, an increase in intermediate type spermatogonia were noted in Bax-deficient animals. By P25, the number of basal compartment cells was greatly increased in Bax-deficient animals compared with controls such that four or five layers of preleptotene spermatocytes were routinely present within the basal compartment of the testis. Although the Sertoli cell barrier was significantly removed from the basement membrane, it appeared intact as judged by the hypertonic fixation test. During late pubertal development, massive degeneration of germ cells took place, including many of those cell types that previously survived in the first wave of spermatogenesis. The data indicate that Bax is required for normal developmental germ cell death in the type A spermatogonia, specifically dividing (A₂, A₃, and A₄) spermatogonia, at a time at which the number of spermatogonia is regulated in a density-dependent manner. The massive hyperplasia that occurs in Bax-deficient mice subsequently results in Bax independent cell death that may be triggered by overcrowding of the seminiferous epithelium.

The purpose of the present study was to examine the effects of progestins on progesterone synthesis and expression of the cytochrome P450 cholesterol side-chain cleavage gene (P450_scc) in a stable porcine granulosa cell line, the JC-410. Cells were incubated for 48 h with the synthetic progestogen-levornorgestrel with or without RU486 (progesterone and glucocorticoid receptor antagonist) or RWJ26819 (progesterone agonist without affinity to glucocorticoid receptors). Both levonorgestrel and RU486 enhanced progesterone accumulation in a dose-dependent manner. RU486 did not antagonize the effects of levonorgestrel, and RWJ26819 had no effect on progesterone production in cultured JC-410 cells. Progesterone and levonorgestrel increased steady state P450_scc mRNA levels after 3–6 h of treatment. Progesterone and RU486 at 0.1, 1, and 10 μM increased the transcription rate of P450_scc transiently expressed in JC-410 cells after 18 h of incubation; 30 μM had no effect, and 100 μM suppressed transcription. Levonorgestrel did not affect transcription of the P450_scc gene, and RWJ26819 reduced its transcription. Progesterone and RU486 significantly decreased the number of cells and total protein content after 72 and 24 h of incubation, respectively. Levonorgestrel had no effect, whereas RWJ26819 increased (24 h) but subsequently reduced (72 h) cell number and protein content. The present results indicate that progestins are capable of directly modulating progesterone biosynthesis in porcine JC-410 granulosa cells. These effects may be exerted in part through the regulation of P450_scc gene expression. Ostensible differences exist between progesterone and its synthetic analogues in the control of progesterone secretion in the stable porcine granulosa cell line in vitro.

Developmental changes in the expression of 18 Leydig cell-specific mRNA species were measured by real-time polymerase chain reaction to partially characterize the developmental phenotype of the cells in the mouse and to identify markers of adult Leydig cell differentiation. Testicular interstitial webs were isolated from mice between birth and adulthood. Five developmental patterns of gene expression were observed. Group 1 contained mRNA species encoding P450 side chain cleavage (P450_scc), P450_c17, relaxin-like factor (RLF), glutathione S-transferase 5-5 (GST5-5), StAR protein, LH receptor, and epoxide hydrolase (EH); group 2 contained 3β-hydroxysteroid dehydrogenase (3β-HSD) VI, 17β-hydroxysteroid dehydrogenase (17β-HSD) III, vascular cell adhesion molecule 1, estrogen sulfotransferase, and prostaglandin D (PGD)-synthetase; group 3 contained patched and thrombospondin 2 (TSP2); group 4 contained 5α-reductase 1 and 3α-hydroxysteroid dehydrogenase; group 5 contained sulfonylurea receptor 2 and 3β-HSD I. Group 1 contained genes that were expressed in fetal and adult Leydig cells and which increased in expression around puberty toward a maximum in the adult. Group 2 contained genes expressed only in the adult Leydig cell population. Group 3 contained genes with predominant fetal/neonatal expression in the interstitial tissue. Group 4 contained genes with a peak of expression around puberty, whereas genes in group 5 show little developmental change in expression. Highest mRNA levels in descending order were RLF, P450_c17, EH, 17β-HSD III, PGD-synthetase, GST5-5, and P450_scc. Results identify five genes expressed in the mouse adult Leydig cell population, but not in the fetal population, and one gene (TSP2) that may be expressed only in the fetal Leydig cell population. The developmental pattern of gene expression suggests that three distinct phases of adult Leydig cell differentiation occur.

The bovine placenta produces estrogens from the first trimester until the end of its life span. However, with the exception of the immediate prepartal and intrapartal phases, in which an involvement of placental estrogens has been suggested for the preparation of parturition, their function has not been elucidated yet. To test for a role of placental estrogens as local factors regulating placental growth and differentiation, placentomes from cows that were pregnant for 150, 220, 240, and 270 days, and parturient cows (3 animals per group) were screened immunohistochemically for the expression of estrogen receptor α (ERα). Indirect immunoperoxidase staining methods were applied using primary monoclonal antibodies (pmAbs) directed against the C-terminus (AER311, HT277) or the N-terminus (AER314, 1D5) of the ERα molecule. Both types of pmAbs identified ERα in stromal cells and capillary pericytes of the maternal caruncular septae. Using pmAb 1D5, the mean percentage of ERα-positive caruncular stromal cells decreased from 39.0% ± 5.9% in pregnant cows to 17.5% ± 8.3% at parturition (P = 0.011). Only pmAb recognizing the C-terminus identified ERα in the caruncular epithelium, in which positive reactions were found in all cells, with the exception of areas adjacent to the chorionic plate and to major chorionic villi, where the specific signal gradually faded and occasionally disappeared. No positive reactions were observed in the fetal part of the placentomes. The expression of ERα in bovine placentomes was further confirmed by the detection of ERα-specific mRNA by reverse transcriptase-polymerase chain reaction and by Western blot analysis. The results suggest a role for placental estrogens as paracrine factors involved in the regulation of placental growth and differentiation.

In the seminiferous epithelium, morphologically diverse junctions mediate inter-Sertoli and Sertoli-germ cell adhesive contact, but the molecular composition of such junctions is not well known. At prototypical adherens junctions, proteins termed catenins bind to the intracellular domain of classic cadherins and regulate the strength of adhesion. Using a panel of monoclonal antibodies (5A7, 8D11, and 15D2), p120 catenin (p120) was localized in postnatal and adult rat testis cryosections and touch preparations by immunofluorescence. Immunoprecipitation of testis homogenates showed that at least four p120 isoforms were expressed from Postnatal Day 7 through adulthood. Both inter-Sertoli and Sertoli-germ cell junctions were p120-positive, however, individual p120 monoclonals were localized to specific junctions. The 5A7 and 8D11 antibodies colocalized with β-catenin and plectin at inter-Sertoli and Sertoli-spermatocyte junctions. At inter-Sertoli junctions, p120 was juxtaposed to but did not colocalize with f-actin. Thus, p120 is likely a component of inter-Sertoli desmosome-like junctions. In contrast, the 15D2 monoclonal antibody specifically immunostained Sertoli-round spermatid and inter-Sertoli cell junctions in a dynamic pattern. From the time that round spermatids form to their differentiation into elongate spermatids, Sertoli-round spermatid 15D2 immunostaining cycled from a single mass to a curvilinear pattern, and finally to punctate structures scattered throughout the epithelium. This localization and stage-specific immunostaining pattern indicated that 15D2 recognized Sertoli-round spermatid desmosome-like junctions. Between Sertoli cells, 15D2 immunostained newly formed junctions (at Postnatal Days 21 through 43), but not mature junctions in the adult. From these data, we conclude that p120 is a component of most, if not all, desmosome-like junctions, and that desmosome-like junctions between different cell types contain a unique molecular composition.

In the seminiferous epithelium, morphologically diverse junctions mediate inter-Sertoli and Sertoli-germ cell adhesive contact and likely transmit signals between contacting cells. Defining the molecular composition of testicular cell-cell junctions is an important step in determining their function. Proteins belonging to the cadherin superfamily are important mediators of cell-cell adhesion, as well as cell signaling. Here, we determined the spatial and temporal protein expression of four classic cadherins in rat testis: N-cadherin, cadherin-6, cadherin-11, and a cadherin defined by an antiserum generated against a conserved classic cadherin peptide (L4). Through Western blot analysis, all antibodies recognized unique proteins. Similarly, each cadherin displayed unique, cell-type specific immunostaining patterns. Whereas N-cadherin, cadherin-11, and L4-positive cadherin were expressed from Postnatal Day 7 through adulthood, cadherin-6 protein was not present at Postnatal Day 7 and first appeared at Day 21. Immunostaining of testis cryosections on Postnatal Days 7, 21, 31, 43, and those of adults indicated that cadherin-11 localized to peritubular cell junctions. N-cadherin immunostaining localized to basal inter-Sertoli junctions, Sertoli-spermatocyte junctions, and at about stages I–VII in Sertoli-elongate spermatid junctions. Cadherin-6 immunostaining was restricted to Sertoli-round spermatid and in Sertoli-elongate spermatid junctions at approximately stages XII–I. Finally, L4-positive immunostaining also detected Sertoli-round spermatid junctions in addition to Sertoli-elongate spermatid junctions at approximately stages XII–I. These data show that the various testicular cell-cell junctions are molecularly unique and dynamic complexes.

Genetically modified domestic animals have many potential applications ranging from basic research to production agriculture. One of the goals in transgenic animal production schemes is to reliably predict the expression pattern of the foreign gene. Establishing a method to screen genetically modified embryos for transgene expression before transfer to surrogates may improve the likelihood of producing offspring with the desired expression pattern. In order to determine how transgene expression may be regulated in the early embryo, we generated porcine embryos from two distinct genetically modified cell lines by using the nuclear transfer (NT) technique. Both cell lines expressed the enhanced green fluorescent protein (eGFP); the first was a fibroblast cell line derived from the skin of a newborn pig that expressed eGFP, whereas the second was a fetal derived fibroblast cell line into which the eGFP gene was introduced by a retroviral vector. The reconstructed embryos were activated by electrical pulses and cultured in NCSU23. Although the in vitro developmental ability of each group of NT embryos was not different, the eGFP expression pattern was different. All embryos produced from the transduced fetal cell line fluoresced, but only 26% of the embryos generated from the newborn cell line fluoresced, and among those that did express eGFP, more than half had a mosaic expression pattern. This was unexpected because the fetal cell line was not clonally selected, and each cell had potentially different sites of integration. Embryos generated from the newborn cell line were surgically transferred to five surrogate gilts. One gilt delivered four female piglets, all of which expressed eGFP, and all had microsatellites identical to the donor. Here we demonstrate that transgene expression in all the blastomeres of an NT embryo is not uniform. In addition, transgene expression in a genetically manipulated embryo may not be an accurate indicator of expression in the resulting offspring.

In preparation for blastocyst implantation, uterine luminal epithelial cells express new cell adhesion molecules on their apical plasma membrane. Since one mechanism epithelial cells employ to regulate membrane polarity is the establishment of specific membrane-cytoskeletal interactions, this study was undertaken to determine if new cytokeratin (CK) intermediate filament assemblies are expressed in endometrial epithelial cells during developmental stages related to blastocyst implantation. Type-specific CK antibodies were used for immunocytochemical and immunoblot analyses of 1) intermediate filament networks of the endometrial epithelium during embryo implantation in rabbits and 2) proliferative and secretory phases of the human menstrual cycle. CK18, a type I CK found in most simple epithelia, was expressed in all luminal and glandular epithelial cells of both the human and rabbit endometrium at all developmental stages analyzed; it was also strongly expressed in trophectoderm of the implanting rabbit blastocyst. In contrast, CK13, another type I cytokeratin, exhibited a regulated expression pattern in luminal, but not glandular, epithelial cells of secretory phase human and peri-implantation stage rabbit endometrium. Furthermore, in the rabbit implantation chambers, CK13 was predominately localized at the cell apex of luminal epithelial cells, where it assembled into a dense filamentous network. These data suggest that the stage-specific expression of CK13 and a reorganization of the apical intermediate filament cytoskeleton of uterine luminal epithelial cells may play important functions in preparation for the implantation process.

Epithelial cells were isolated from adult porcine vas deferens and grown in the absence or presence of steroid hormones. Transepithelial resistance (R_te), basal short circuit current (I_sc), and the effects of selected ion transport modulators on these parameters were evaluated in modified Ussing chambers at three time points (5–8, 11–14, and 18–22 days postseeding). At the earliest time point, no significant differences were observed. At the middle time point, when compared with R_te in untreated control monolayers, R_te in monolayers exposed to 17β-estradiol, aldosterone, cortisol, cortisone, prednisolone, prednisone, and dexamethasone was significantly lower; in contrast, R_te in monolayers exposed to testosterone, dihydrotestosterone, or progesterone did not differ from that in control monolayers. Treatments with cortisol, prednisolone, and dexamethasone were associated with an elevated basal I_sc that was amiloride sensitive, indicating ongoing Na absorption by these monolayers. R_te was increased by amiloride treatment in glucocorticoid-treated monolayers but remained significantly less than that of control monolayers. At the third time point, the postamiloride R_te of glucocorticoid-treated monolayers was not different from that of control monolayers. Responses to ATP, forskolin, bumetanide, and DASU-02 were not affected by steroid treatment at any time point. Taken together, these results suggest that estrogens and corticosteroids can modulate epithelial function in the distal excurrent duct of the adult male reproductive system. At physiological or pharmacological concentrations, these hormones would be expected to modify the luminal environment (both the ionic composition and pH) to which sperm are exposed and thus affect male fertility.

Increases in the survival rate of men treated with chemotherapeutic drugs and their desire to have children precipitate concerns about the effects of these drugs on germ cells. Azoospermia, oligospermia, and infertility are common outcomes resulting from treatment with cyclophosphamide, an alkylating agent. Exposure of male rats to cyclophosphamide results in dose-dependent and time-specific adverse effects on progeny outcome. Elucidation of the effects of chronic low-dose cyclophosphamide treatment on the expression of stress response genes in male germ cells may provide insight into the mechanisms underlying such adverse effects. Male rats were gavaged with saline or cyclophosphamide (6 mg/kg) for 4–5 wk; pachytene spermatocytes, round spermatids, and elongating spermatids were isolated; RNA was extracted and probed on cDNA arrays containing 216 cDNAs. After saline treatment, 125 stress response genes were expressed in pachytene spermatocytes (57% of genes studied), 122 in round spermatids (56%), and 83 in elongating spermatids (38%). Cyclophosphamide treatment reduced the number of genes detected in all germ cell types. The predominant effect of chronic cyclophosphamide exposure was to decrease the expression level of genes in pachytene spermatocytes (34% of genes studied), round spermatids (29%), and elongating spermatids (4%). In elongating spermatids only, drug treatment increased the expression of 8% of the genes studied. The expression profiles of genes involved in DNA repair, posttranslational modification, and antioxidant defense in male germ cells were altered by chronic cyclophosphamide treatment. We hypothesize that the effects of cyclophosphamide exposure on germ cell gene expression during spermatogenesis may have adverse consequences on male fertility and progeny outcome.

Porcine in vitro production (IVP) systems, including in vitro maturation (IVM) and in vitro fertilization (IVF) of oocytes and their subsequent in vitro culture (IVC), have been modified by many researchers, but are still at a low level because of a low developmental rate of embryos to the blastocyst stage and their poor qualities. Our objectives were to establish reliable IVP procedures for porcine blastocysts and to examine the ability of the blastocysts to develop to term after transfer to recipients. Porcine cumulus-oocyte complexes were matured in vitro under 5% O₂ or 20% O₂, fertilized in vitro under 5% O₂, and subsequently cultured under 5% O₂ in 1) IVC medium supplemented with glucose (IVC-Glu) from Day 0 (the day of IVF) to Day 6; 2) IVC-Glu from Days 0 to 2, then IVC medium supplemented with pyruvate and lactate (IVC-PyrLac) from Days 2 to 6; 3) IVC-PyrLac from Days 0 to 2, then IVC-Glu from Days 2 to 6; and 4) IVC-PyrLac from Days 0 to 6. There were no significant differences in blastocyst formation rates on Day 6 between the 5% O₂ and 20% O₂ conditions (19.9% and 14.0%, respectively). However, the quality of blastocysts, as evaluated by the total cell number, was better after IVM under 5% O₂ than under 20% O₂ (mean cell number, 43.5 and 37.8, respectively). When IVP embryos were cultured in IVC-PyrLac from Days 0 to 2 and subsequently in IVC-Glu from Days 2 to 6, the rate of blastocyst formation (25.3%) and cell number (48.7) were higher than the rates (5.8% to 18.1%) and numbers (35.4 to 37.1) with the IVC-Glu then IVC-Glu, the IVC-Glu then IVC-PyrLac, and the IVC-PyrLac then IVC-PyrLac regimens, respectively. We then prepared conditioned medium (CM) from culture of porcine oviductal epithelial cells for 2 days in IVC-PyrLac and evaluated its effect on development to the blastocyst stage. Cultivation in CM for the first 2 days, followed by IVC-Glu for a further 4 days, had a significantly greater effect in increasing the number of cells in the blastocyst (58.3) than did in IVC-PyrLac (48.4). Finally, we evaluated the ability of blastocysts, generated by IVM under 5% O₂ and IVC in CM, to develop to term. When Day 5 expanding blastocysts (mean cell number, 49.7) were transferred to an estrus-synchronized recipient (50 blastocysts per recipient), the recipient remained pregnant and farrowed eight normal piglets. Furthermore, when Day 6 expanded blastocysts (mean cell number, 80.2) were transferred to two estrus-synchronized recipients, both gilts remained pregnant and farrowed a total of 11 piglets. These results suggest that an excellent piglet production system can be established by using this modified IVP system, which produces high-quality porcine blastocysts. This system has advantages for the generation of cloned and transgenic pigs.

The secretion and localization of clusterin in the testis has led to the hypothesis that clusterin plays a role in spermatogenesis. Furthermore, the association of clusterin with apoptosis, cellular injury, disease, and regression of nongonadal tissues has led to the hypothesis that clusterin acts to protect cells from apoptosis or may be involved in tissue remodeling. To investigate the role of clusterin in the testis, we analyzed clusterin knock-out (cluKO) mice to determine the impact of the absence of clusterin on spermatogenesis. Furthermore, we investigated the cellular response to injury caused by methoxyacetic acid (MAA) toxicity and mild heat exposure in the cluKO mice to determine the extent to which clusterin protects against apoptosis or participates in tissue remodeling. We found that cluKO mice were fertile and had essentially normal spermatogenesis with the exception of some incomplete spermiation after stage VIII. No differences in testicular morphology or the incidence of apoptosis in the testis were seen between the cluKO and clusterin wild-type (cluWT) mice after MAA treatment. In contrast, apoptosis was delayed in the cluWT mice compared with the cluKO mice after heat exposure, suggesting that clusterin does have a slight protective effect against apoptosis under some conditions. Also, a dramatic loss of germ cells after heat stress occurred earlier in the cluWT testes than in the cluKO testes. Clusterin is clearly acting in a dual role in that cells can be protected from damage and dead cells can be more easily removed after some types of cellular damage but not after others.

In adult mammals, estrogen regulates ovarian function, and estrogen receptor (ER) is expressed in granulosa cells of antral follicles of the adult baboon ovary. Because the foundation of adult ovarian function is established in utero, the present study determined whether ERα and/or ERβ were expressed in fetal ovaries obtained on Days 100 (n = 3) and 165–181 (n = 5) of baboon gestation (term = Day 184). On Day 100, ERα protein was detected by immunocytochemistry in surface epithelium and mesenchymal-epithelial cells but not oocytes in germ cell cords. ERβ protein was also detected by immunocytochemistry on Day 100 of gestation and was abundantly expressed in mesenchymal-epithelial cells in germ cell cords, lightly expressed in the germ cells, but was not detected in the surface epithelium. On Days 165–180 of gestation, ERα expression was still intense in the surface epithelium, in mesenchymal-epithelial cells throughout the cortex, and in nests of cells between follicles. ERα expression was lighter in granulosa cells and was not observed in all granulosa cells, particularly in follicles close to the cortex. In contrast, ERβ expression was most intense in granulosa cells, especially in flattened granulosa cells, was weaker in mesenchymal-epithelial cells and nests of cells between follicles, and was absent in the surface epithelium. Using an antibody to the carboxy terminal of human ERβ, ERβ protein was also detected by Western immunoblot with molecular sizes of 55 and 63 kDa on Day 100 and primarily 55 kDa on Day 180. The mRNAs for ERα and ERβ were also detected by Northern blot analysis in the baboon fetal ovary. These results are the first to establish that the ERα and ERβ mRNAs and proteins are expressed and exhibit changes in localization in the primate fetal ovary between mid and late gestation. Because placental estrogen production and secretion into the baboon fetus increases markedly during advancing pregnancy, we propose that estrogen plays an integral role in programming fetal ovarian development in the primate.

Numerous studies have shown the presence of DNA strand breaks in human ejaculated spermatozoa. The nature of this nuclear anomaly and its relationship to patient etiology is however poorly understood. The aim of this study was to investigate the relationship between nuclear DNA damage, assessed using the TUNEL assay and a number of key apoptotic markers, including Fas, Bcl-x, and p53, in ejaculated human spermatozoa from men with normal and abnormal semen parameters. We also determined the nature of the DNA damage by examining the percentage of ejaculated spermatozoa exhibiting DNA damage using the comet assay and by challenging sperm chromatin to attack by micrococcal nuclease S7 and DNase I. We show that TUNEL positivity and apoptotic markers do not always exist in unison; however, semen samples that had a low sperm concentration and poor morphology were more likely to show high levels of TUNEL positivity and Fas and p53 expression. In addition, the DNA damage in ejaculated human sperm is represented by both single- and double-stranded DNA breaks, and access to the DNA is restricted by the compacted nature of ejaculated spermatozoa. This DNA protection is poorer in men with abnormal semen parameters. We propose that the presence of DNA damage is not directly linked to an apoptotic process occurring in spermatozoa and arises due to problems in the nuclear remodeling process. Subsequently, the presence of apoptotic proteins in ejaculated spermatozoa may be linked to defects in cytoplasmic remodeling during the later stages of spermatogenesis.

In turkey hens, the egg production rate is relatively high early during a reproductive period, but declines as the period progresses. Among lines with different egg production potential, the interval between preovulatory surges of LH is the primary determinant of the egg production rate. The main objective of this study was to determine whether the decline in egg production rate late during an egg production period is also associated with a difference in the interval between LH preovulatory surges. A group of photosensitive turkey hens (Early) were photostimulated with continuous light (24L:0D) at 40 wk of age to induce egg laying, and serial blood samples were collected after about 3 wk of egg production. A second group of hens (Late) were housed in floor pens and photostimulated with 14L:10D at 40 wk of age for a normal 36-wk reproduction period and were then switched to 24L:0D lighting for 2 wk before collection of serial blood samples. Continuous light photostimulation was used for at least 2 wk before and during serial blood sampling to avoid potential masking effects of diurnal lighting on the interval between LH surges. The Early (n = 12) and Late (n = 16) hens were cannulated 3 days before being serially bled hourly for 10 days. The mean interval between preovulatory surges of LH was shorter in the Early hens than in the Late hens (26.1 ± 2.5 h and 34.7 ± 3.9 h, respectively). The intra-hen LH surge interval coefficient of variation was lower in the Early hens than in the Late hens (7.2% and 18.6%, respectively). The inter-hen LH surge interval coefficient of variation was similar in the Early and Late hens (9.5% and 11.2%, respectively). The incidence of blind surges of LH (those not retrospectively associated with ovipositions) was not different between Early and Late laying hens (8.4% ± 15.2% and 7.3% ± 14.6%, respectively). In conclusion, in turkey hens, longer intervals and greater intra-hen variation between LH surges were associated with a poorer rate of egg production late in the reproductive period relative to early in the reproductive period.

The lipolytic enzyme hepatic lipase (HL) may facilitate mobilization of cholesterol substrate for ovarian steroidogenesis. We investigated whether HL was necessary for optimum reproduction in the female mouse by analyzing breeding performance and ovarian responses to gonadotropins in HL−/− mice. HL−/− female mice bred with HL−/− males had the same pregnancy success rate and pup survival rate as did wild-type (WT) mice but had significantly smaller litters, producing 1.7 fewer pups per litter. Mice were primed with eCG/hCG, and at 6 h post-hCG the HL−/− mice had smaller ovaries than did the WT mice. HL deficiency specifically affected ovarian weight; adrenal gland weights did not differ between WT and HL−/− mice. HL−/− mice weighed more than age-matched WT mice. Between the two mouse genotypes, uterine weights were the same, indicating that estrogen production was equivalent. However, the HL−/− ovaries produced significantly less progesterone than did the WT ovaries within 6 h of hCG stimulation. HL−/− ovaries had the same number of large antral follicles as did the WT ovaries but had fewer hemorrhagic sites, which represent ovulations, fewer corpora lutea, and more oocytes trapped in corpora lutea. We suggest that reduced progesterone synthesis following hCG stimulation attenuated the final maturation of preovulatory follicles, resulting in smaller ovaries. Furthermore, reduced progesterone production limited the expression of proteolytic enzymes needed for tissue remodeling, resulting in fewer ovulations with a corresponding increase in trapped or unovulated oocytes and providing a possible explanation for the smaller litter size observed in spontaneously ovulating HL−/− mice.

Members of the matrix metalloproteinase (MMP) family collectively degrade extracellular matrix (ECM) and help regulate luteal function. The objectives of these experiments were to characterize the mRNA expression, localization, and activity of MMPs 2, 9, and 14 in ovine corpora lutea (CL). Ovine CL were collected on Days 2, 4, 10, and 15 of the estrous cycle (Day 0 = estrus). Messenger RNA transcripts for MMPs 2 and 14 were detected using Northern analysis; however, expression of MMP-9 was undetectable. Expression of MMP-14 mRNA (membrane type-1 MMP) was increased (P < 0.05) on Day 4; whereas, expression of MMP-2 mRNA was highest (P < 0.05) on Day 10, which corresponded to the observed increases in gelatinolytic activity in luteal homogenates as measured by a fluroscein-labeled gelatin substrate assay. MMP 2 and 9 proteins were localized predominantly to large luteal cells (LLCs), whereas MMP-14 was localized primarily to cells other than LLCs as demonstrated by immunohistochemistry. Immunolocalization of MMP-2 to putative endothelial cells was also observed on Day 15. Localization of MMP activity was determined using in situ zymography. Luteal tissues contained gelatinolytic activity primarily localized pericellularly to various cell types, including LLCs. These results support the hypothesis that ECM remodeling occurs throughout the luteal phase and may help potentiate cellular migration, differentiation, angiogenesis, and growth factor bioavailability.

A study was undertaken to determine whether injection of porcine sperm factors (pSF), which trigger oscillations in intracellular calcium concentration ([Ca² ]_i) in mammalian oocytes, could be used to activate bovine oocytes during nuclear transfer. To date, only combined treatments that induce a monotonic rise in [Ca² ]_i and inhibit either phosphorylation or protein synthesis have been utilized in nuclear transfer. Several doses of pSF were tested. Injection of 5 mg/ml pSF triggered [Ca² ]_i oscillations that resembled those associated with fertilization with respect to amplitude and periodicity, and as a result, a high percentage of oocytes underwent activation. Furthermore, this concentration of pSF supported in vitro and in vivo development up to 60–90 days of gestation, comparable to development in control nuclear transfer embryos. Nevertheless, neither activation procedure supported development as well as did fertilization. The effectiveness of pSF as an activating agent in bovine oocytes may have been compromised because pSF was unable to support oscillations past 3–5 h postinjection and a second injection was necessary to extend the [Ca² ]_i oscillations. Likewise, a single injection of pSF failed to trigger downregulation of the inositol 1,4,5-trisphosphate receptor 1 subtype, whereas a second injection downregulated the receptor in a manner similar to that seen in fertilized oocytes. These results demonstrate that soluble factor(s) from porcine sperm can support early development in bovine nuclear transfer embryos; however, the efficacy may be limited because of the premature cessation of the induced oscillations.

Chronic exposure of young ovariectomized rats to elevated circulating estradiol causes loss of steroid-induced LH surges. Such LH surges are associated with cFos-induced activation of GnRH neurons; therefore, we hypothesized that chronic estradiol treatment abolishes LH surges by decreasing activation of GnRH neurons. Regularly cycling rats were ovariectomized and immediately received an estradiol implant or remained untreated. Three days or 2 or 4 wk later, the estradiol-treated rats received vehicle or progesterone at 1200 h, and 7 hourly blood samples were collected for RIA of LH. Thereafter, all rats were perfused, and the brains were examined for immunocytochemical localization of cFos and GnRH. The GnRH neurons from untreated ovariectomized rats rarely expressed cFos. As reported, LH surges induced by 3 days of estradiol treatment were associated with a 30% increase in cFos-containing GnRH neurons, and progesterone enhanced both the amplitude of LH surges and the proportion of cFos-immunopositive GnRH neurons. As hypothesized, the abolition of LH surges caused by 2 or more weeks of estradiol was paralleled by a reduction in the percentage of cFos-containing GnRH neurons, and this effect was delayed by progesterone. These results suggest that chronic estradiol abolishes steroid-induced LH surges in part by inactivating GnRH neurons.

The temporal relationships in the changes in concentrations of follicular fluid factors during follicle selection were characterized in mares. All follicles ≥5 mm were ablated 10 days after ovulation, followed by follicular fluid collection from the three largest follicles (F1, F2, and F3) when F1 of the new wave reached a diameter of 8.0–11.9, 12.0–15.9, 16.0–19.9, 20.0–23.9, 24.0–27.9, or 28.0–31.9 mm (n = 4–8 mares/range). Diameter deviation between F1 and F2 began during the 20.0- to 23.9-mm range, as indicated by a greater difference in diameter between the two follicles at the 24.0- to 27.9-mm range than at the 20.0- to 23.9-mm range. Androstenedione concentrations increased in F1, F2, and F3 between the 16.0- to 19.9- and 20.0- to 23.9-mm ranges. In contrast, estradiol, free insulin-like growth factor (IGF)-1, activin-A, and inhibin-A concentrations increased only in F1 beginning at the 16.0- to 19.9-mm range. As a result, the concentrations of all four factors were higher in F1 than in F2 and F3 at all the later ranges, including the 20.0- to 23.9-mm range (beginning of diameter deviation). Concentrations of progesterone differentially increased in F1, concentrations of androstenedione and IGF-binding protein (IGFBP)-2 increased only in F2 and F3, and concentrations of inhibin-B differentially decreased in F2 and F3 simultaneous with the beginning of deviation. Concentrations of FSH, LH, pro-αC inhibin, and total inhibin did not change differentially among follicles. Results indicated that, on a temporal basis, estradiol, free IGF-1, activin-A, and inhibin-A may have played a role in the initiation of follicle deviation. In addition, these four factors as well as progesterone, androstenedione, IGFBP-2, and inhibin-B may have been involved in the subsequent differential development of the follicles.

Quantitative changes in ovarian inhibin/activin subunit and follistatin mRNAs during the rat estrous cycle were examined by ribonuclease protection assay using digoxygenin-labeled RNA probes. Levels of ovarian inhibin α subunit mRNA remained low throughout estrus, metestrus, and diestrus; abruptly increased on the morning of proestrus; then rapidly decreased when the primary gonadotropin surge occurred. A similar changing pattern was observed in inhibin/activin β_A subunit mRNA. On the other hand, inhibin/activin β_B subunit mRNA showed a different changing pattern. Levels of β_B subunit mRNA remained constant during metestrus and diestrus, abruptly decreased on the afternoon of proestrus, then quickly recovered from the nadir by 1100 h on estrus. Throughout the rat estrous cycle, especially during the periovulatory period, α subunit mRNA levels were considerably higher than β_A and β_B subunit mRNA levels. In addition, changes in plasma concentrations of inhibin A and inhibin B were very similar to that in ovarian β_A and β_B subunit mRNA levels, respectively, with several-hour delays. These results suggest that levels of β subunit mRNAs restrict secretion of dimeric inhibins. Levels of follistatin mRNA remained low from the midnight of metestrus to the midnight of diestrus, then increased until initiation of the primary gonadotropin surge. Thereafter, follistatin mRNA decreased, reached the nadir at 0200 h on estrus, then increased abruptly at 1100 h on estrus. Afterward, follistatin mRNA levels remained high until the morning of metestrus. The changing pattern of ovarian follistatin mRNA was similar to, and preceded, the changes in plasma concentrations of progesterone, suggesting that ovarian follistatin may modulate progesterone secretion during the rat estrous cycle.

The zebra finch (Taeniopygia guttata) brain is highly sexually dimorphic. The organization and production of sex-specific song is considerably influenced by estrogens and androgens. Because the brain itself expresses several steroidogenic enzymes, the local production of sex steroids may contribute to sex differences in neural development. Sex steroid production in gonads is directed by a master regulatory factor, steroidogenic factor 1 (SF1). We have identified a cDNA encoding the homologue of SF1 in the zebra finch and utilized reverse transcription-polymerase chain reaction and in situ hybridization to examine early and late developmental expression of SF1 in brain and in early gonadal development. We found that SF1 is expressed early in embryonic development in the Rathke pouch, beginning at stage 15 and extending to at least stage 27 in both males and females. The earliest expression of SF1 in gonads was found at stage 17 for both males and females and extended to at least stage 27. In brain, we assessed SF1 mRNA expression in posthatch and adult telencephalon, and we compared SF1 and aromatase mRNA expression in adult hypothalamus. In the telencephalon and hippocampus, aromatase was expressed independently of SF1, whereas in the hypothalamus, aromatase and SF1 expression were more closely associated. Expression of SF1 and of aromatase overlapped in restricted regions of the hypothalamus, suggesting that SF1 may regulate aromatase expression in these regions. These findings suggest that steroidogenesis in the zebra finch brain may be regulated by both SF1-dependent and SF1-independent mechanisms. No sex differences were detected in SF1 expression in brain.

The origin of follicle (i.e., pregranulosa) cells that become the somatic component of primordial follicles is obscure. In addition, information regarding the structural changes that accompany the concomitant regression of ovigerous cords and the appearance of primordial follicles is lacking. In the present study, ovine ovaries collected at frequent time intervals between Day 38 and Day 100 of fetal life were examined by light and electron microscopy. To gain new information regarding the origin of follicular cells, incorporation of 5-bromo-2′-deoxyuridine was used to identify proliferating cells at selected stages of development. Based on the location and identity of proliferating cells, apoptotic cells, and sequential changes in histoarchitecture, we hypothesize 1) that most (i.e., >95%) of the granulosal cells in newly formed primordial follicles originate from the ovarian surface epithelium; 2) that the sequential events leading to follicle formation take place entirely within ovigerous cords, with the first follicles forming at the interface of the cortex and medulla; and 3) that the loss (i.e., >75%) of germ cells, but not of somatic cells, within the ovigerous cords is a means by which each surviving oocyte gains additional pregranulosal cells before follicle formation. Conceptual models detailing the chronology of developmental events involved in the formation of primordial follicles in sheep are discussed.

In humans, follicle quantity and quality decline with age by atresia. In the present study we aimed to describe the quality of the follicle pool through an ultrastructural investigation of resting follicles in young healthy women. From ovarian biopsies of 7 women aged 25–32 yr, 182 small follicles were morphometrically assessed for various signs of atresia. Morphometric variables were analyzed by principal components analysis (PCA) to demonstrate correlations between variables and to construct an objective follicle score. One third of small follicles consisted of primordial follicles. Nucleus:cell ratios remained constant for oocytes and granulosa cells from primordial to primary follicles, suggesting that follicles up to primary stages belong to the resting pool. The distribution of follicle quality scores as derived from PCA showed that most follicles were of good quality and with little signs of atresia. Atresia in resting follicles appears to be a necrotic process, starting in the ooplasma. Early atresia was characterized by increasing numbers of multivesicular bodies and lipid droplets, dilation of smooth endoplasmic reticulum and Golgi, and irregular mitochondria with changed matrix density. In progressive atresia mitochondrial membranes ruptured, oocyte nuclear membranes were indented or ruptured, and the ooplasma showed extensive vacuolarization. The early involvement of mitochondria in this process suggests that damage is induced by oxygen radicals. PCA follicle quality scores can be reliably approximated using a reduced number of seven morphometric variables, which were selected by stepwise forward analysis. The algorithm to calculate these follicle scores is presented.

Expression of estrogen receptors (ERs) in the reproductive tracts of adult male dogs and cats has not been reported. In the present study, ERα and ERβ were localized by immunohistochemistry using ER-specific antibodies. ERα was found in interstitial cells and peritubular myoid cells in the dog testis, but only in interstitial cells of the cat. In rete testis of the dog, epithelial cells were positive for ERα staining, but in the cat, rete testis epithelium was only weakly positive. In efferent ductules of the dog, both ciliated and nonciliated cells stained intensely positive. In the cat, ciliated epithelial cells were less stained than nonciliated epithelial cells. Epithelial cells in dog epididymis and vas deferens were negative for ERα. In the cat, except for the initial region of caput epididymis, ERα staining was positive in the epithelial cells of epididymis and vas deferens. Multiple cell types of dog and cat testes stained positive for ERβ. In rete testis and efferent ductules, epithelial cells were weakly positive for ERβ. Most epithelial cells of the epididymis and vas deferens exhibited a strong positive staining in both species. In addition, double staining was used to demonstrate colocalization of both ERα and ERβ in efferent ductules of both species. The specificity of antibodies was demonstrated by Western blot analysis. This study reveals a differential localization of ERα and ERβ in male dog and cat reproductive tracts, demonstrating more intensive expression of ERβ than ERα. However, as in other species, the efferent ductules remained the region of highest concentration of ERα.

Apoptosis is a form of cell death that can function to eliminate cells damaged by environmental stress. One stress that can compromise embryonic development is elevated temperature (i.e., heat shock). For the current studies, we hypothesized that heat shock induces apoptosis in bovine embryos in a developmentally regulated manner. Studies were performed to 1) determine whether heat shock can induce apoptosis in preimplantation embryos, 2) test whether heat-induced apoptosis is developmentally regulated, 3) evaluate whether heat shock-induced changes in caspase activity parallel patterns of apoptosis, and 4) ascertain whether exposure to a mild heat shock can protect embryos from heat-induced apoptosis. As determined by TUNEL reaction, exposure of bovine embryos ≥16 cells on Day 5 after insemination to 41 or 42°C for 9 h increased the percentage of cells undergoing apoptosis. In addition, there was a duration-dependent increase in the proportion of blastomeres that were apoptotic when embryos were exposed to temperatures of 40 or 41°C, which are more characteristic of temperatures experienced by heat-stressed cows. Heat shock also increased caspase activity in Day 5 embryos. However, heat shock did not induce apoptosis in 2- or 4-cell embryos, nor did it increase caspase activity in 2-cell embryos. The apoptotic response of 8- to 16-cell-stage bovine embryos to heat shock depended upon the day after insemination that heat shock occurred. When 8- to 16-cell embryos were collected on Day 3 after insemination, heat shock of 41°C for 9 h did not induce apoptosis. In contrast, when 8- to 16-cell embryos were collected on Day 4 after insemination and exposed to heat shock, there was an increase in the percentage of cells undergoing apoptosis. Exposure of 8- to 16-cell embryos at Day 4 to a mild heat shock of 40°C for 80 min blocked the apoptotic response to a subsequent, more-severe heat shock of 41°C for 9 h. In conclusion, apoptosis is a developmentally acquired phenomenon that occurs in embryos exposed to elevated temperature, and it can be prevented by induced thermotolerance.

Cloning by somatic cell nuclear transfer is critically dependent upon early events that occur immediately after nuclear transfer, and possibly additional events that occur in the cleaving embryo. Embryo culture conditions have not been optimized for cloned embryos, and the effects of culture conditions on these early events and the successful initiation of clonal development have not been examined. To evaluate the possible effect of culture conditions on early cloned embryo development, we have compared a number of different culture media, either singly or in sequential combinations, for their ability to support preimplantation development of clones produced using cumulus cell nuclei. We find that glucose is beneficial during the 1-cell stage when CZB medium is employed. We also find that potassium simplex optimized medium (KSOM), which is optimized to support efficient early cleavage divisions in mouse embryos, does not support development during the 1-cell or 2-cell stages in the cloned embryos as well as other media. Glucose-supplemented CZB medium (CZB-G) supports initial development to the 2-cell stage very well, but does not support later cleavage stages as well as Whittten medium or KSOM. Culturing cloned embryos either entirely in Whitten medium or initially in Whittens medium and then changing to KSOM at the late 4-cell/early 8-cell stage produces consistent production of blastocysts at a greater frequency than using CZB-G medium alone. The combination of Whitten medium followed by KSOM resulted in an increased number of cells per blastocyst. Because normal embryos do not require glucose during the early cleavage stages and develop efficiently in all of the media employed, these results reveal unusual culture medium requirements that are indicative of altered physiology and metabolism in the cloned embryos. The relevance of this to understanding the kinetics and mechanisms of nuclear reprogramming and to the eventual improvement of the overall success in cloning is discussed.

Specialized natural killer (NK) lymphocytes are a feature of the pregnant uterus in humans and rodents. Conceptus-mediated recruitment of uterine (u)NK cells in the pig was proposed based on evidence that elevated uNK activity was temporally associated with increased leukocyte density in endometrium underlying conceptuses. The objective of this study was to determine whether uNK cells were more abundant at embryonic attachment sites during the early postattachment period. Mononuclear leukocytes were isolated from endometrium at attachment sites versus between attachment sites, and expression of CD16, a marker for NK cells, was assessed by flow cytometry. CD16 binding was normalized to leukocyte numbers in each sample. CD16 small lymphocytes were more frequent in uterus than in blood (41% ± 2% versus 26% ± 4%). Differences between pregnant and luteal phase uterus (43% ± 2% versus 31% ± 7%, respectively) were not statistically significant. In pregnant animals, CD16 lymphocytes were slightly but significantly more abundant in uterus at attachment sites versus between attachment sites at Days 15–17, 21–22, and 25–28. Before normalization, CD16 large, granular cells were more abundant at attachment sites versus between attachment sites; however, these differences were removed when data were normalized according to leukocyte numbers. Further characterization showed that the proportion of large granular leukocytes expressing CD8, reactive with NK cells and T cell subsets, was 2-fold higher in pregnant uterus than in maternal blood. These results raise the possibility that uNK cells resembling those in blood may be transformed into larger, more granulated forms in the uterine microenvironment.

Fertilin β (also known as ADAM2), a mammalian sperm protein that mediates gamete cell adhesion during fertilization, is a member of the ADAM protein family whose members have disintegrin domains with homology to integrin ligands found in snake venoms. Fertilin β utilizes an ECD sequence within its disintegrin domain to interact with the egg plasma membrane; the Asp is especially critical. Based on what is known about different integrin subfamilies and their ligands, we sought to characterize fertilin β binding sites on mouse eggs, focusing on integrin subfamilies that recognize short peptide sequences that include an Asp residue: the α₅/α₈/α_v/α_IIb or RGD-binding subfamily (α₅β₁, α₈β₁, α_Vβ₁, α_Vβ₃, α_Vβ₅, α_Vβ₆, α_Vβ₈, and α_IIbβ₃) and the α₄/α₉ subfamily (α₄β₁, α₉β₁, and α₄β₇). We tested peptide sequences known to perturb interactions mediated by these integrins in two different assays for fertilin β binding. Peptides with the sequence MLDG, which perturb α₄/α₉ integrin-mediated interactions, significantly inhibit fertilin β binding to eggs, which suggests a role for a member of this integrin subfamily as a fertilin β receptor. RGD peptides, which perturb α₅/α₈/α_v/α_IIb integrin-mediated interactions, have partial inhibitory activity. The anti-α₆ antibody GoH3 has little or no inhibitory activity. An antibody to the integrin-associated tetraspanin protein CD9 inhibits the binding of a multivalent presentation of fertilin β (immobilized on beads) but not soluble fertilin β, which we speculate has implications for the role of CD9 in the strengthening of fertilin β-mediated cell adhesion but not in initial ligand binding.

Vitelline envelopes (VEs) of Bufo arenarum were isolated in order to study their composition and their role in fertilization. VEs are composed of four glycoproteins, with molecular masses of 120, 75, 41, and 38 kDa. To characterize its biological properties, we quantitatively determined sperm-VE binding and the induction of the acrosome reaction. Heterologous binding of B. arenarum sperm to Xenopus laevis VE components was observed with about one-third the efficiency of homologous binding. Equivalent binding of X. laevis sperm to the B. arenarum VE was observed. When B. arenarum sperm were incubated with fluorescein isothiocyanate-labeled VE, the labeled glycoproteins bound to the anterior end of the sperm head, showing a lateral distribution. Induction of the acrosome reaction was evaluated by incubating sperm in hypotonic saline media with VE glycoproteins. VEs induced the acrosome reaction in a time- and concentration-dependent manner. The acrosome reaction was maximal after 10 min. The half-maximal effect was obtained at a glycoprotein concentration of 1 μg/ml. Specificity was determined using fertilization envelope glycoproteins, which failed to induce the acrosome reaction. The B. arenarum VE is biochemically similar to other egg envelopes. It also seems that its biological properties are similar to other species in regard to sperm binding and induction of the acrosome reaction. However, as far as we are aware, this is the first observation of the VE inducing the sperm acrosome reaction in amphibians. The relatively small differences observed in heterologous sperm-VE binding in X. laevis and B. arenarum are inconsistent with the current paradigm that species specificity in fertilization is regulated at the sperm-VE binding step.

Prolactin (PRL) exerts pleiotropic physiological effects in various cells and tissues, although it is mainly considered as a regulator of reproduction and cell growth. Null mutation of the prolactin receptor (PRLR) gene leads to female sterility due to a failure of embryo implantation. Using this mouse model and the method of mRNA differential display, we identified PRL target genes that are regulated during the peri-implantation period. We characterized 1 among the 45 isolated genes, UA-3, which is regulated in the uterus as well as in the ovary during early pregnancy. This gene corresponds to a P311 mouse cDNA that was originally identified for its high expression in late-stage embryonic brain and adult cerebellum. We report here that UA-3 is present in numerous tissues as well as in ovary and uterus at the site of blastocyst apposition, and that its expression is hormonally regulated. Moreover, in situ hybridization reveals high expression in ovarian granulosa cells and in uterine epithelium. Recently, it has been suggested that P311 expression is tightly regulated at several levels by mechanisms that control cellular growth, transformation, motility, or a combination of these. Taken together, these results suggest that P311 could be involved in these processes during pregnancy, although its function remains to be clearly established.