The cadherins and their cytoplasmic counterparts, the catenins, form the adherens junctions, which are of importance for tissue integrity and barrier functions. The development and maturation of the ovarian follicle is characterized by structural changes, which require altered expression or function of the components involved in cell-cell contacts. The present study examined the cell-specific localization and temporal expression of epithelial cadherin (E-cadherin) and alpha- and beta-catenin during follicular development, ovulation and corpus luteum formation in the immature gonadotrophin- and oestrogen-stimulated rat ovary. Immunohistochemistry and immunoblotting demonstrated the expression of E-cadherin in theca and interstitial cells of immature ovaries before and after injection of equine chorionic gonadotrophin (eCG). E-cadherin was not detected in granulosa cells, except in the preantral follicles located to the inner region of the ovary. The content of E-cadherin in theca and interstitial cells decreased after an ovulatory dose of hCG. Granulosa cells of apoptotic follicles did not express E-cadherin. Oestrogen treatment (diethylstilboestrol) of immature rats for up to 3 days did not result in a measurable expression of E-cadherin in granulosa cells. alpha- and beta-catenin were expressed in all ovarian compartments. The concentration of beta-catenin was constant during the follicular phase, whereas the content of alpha-catenin decreased in granulosa cells after treatment with diethylstilboestrol or hCG. The expression of alpha-catenin was also reduced in theca and interstitial cells after hCG. alpha- and beta-catenin were present in most ovarian cells at all stages of folliculogenesis. Therefore, the catenins have the potential to associate with different members of the cadherin family and to participate in the regulation of cytoskeletal structures and intracellular signalling. The restricted expression of E-cadherin in granulosa cells of preantral follicles indicates a role in the recruitment of these follicles to subsequent cycles. The specific decrease of alpha-catenin in granulosa cells and the reduction of both alpha-catenin and E-cadherin in theca cells of ovulatory follicles might reflect some of the molecular changes in cell-cell adhesion associated with ovulation and luteinization.
The objective of this study was to develop a model for the study of abnormal ovarian follicles in cattle by treating heifers with adrenocorticotrophic hormone (ACTH) (100 iu at 12 h intervals for 7 days, beginning on day 15 of the oestrous cycle). Cortisol concentrations increased (P < 0.05) within 24 h after beginning ACTH treatment and cortisol and progesterone concentrations remained elevated after cessation of ACTH treatment for 8 and 4 days, respectively. The pulses and surges of LH decreased during ACTH treatment, but FSH profiles were similar to those in controls and persistent or prolonged follicles were eventually observed in all heifers. In five heifers, prolonged dominant follicles ovulated after 10 days, whereas in six heifers, persistent follicular structures were present for 20 days, but ceased to secrete oestradiol after approximately 12 days. In the heifers with persistent follicular structures, new follicles emerged when the persistent follicle became non-oestrogenic. During the last 2 days of normal follicular growth, the concentration of oestradiol was greater than it was during prolonged or persistent follicle development (P < 0.05). There were no differences in the growth rates or maximum diameters of abnormal follicles that had different outcomes, but oestradiol concentrations were greater in prolonged follicles that ovulated compared with those follicles that persisted (P = 0.06). In conclusion, stimulation with ACTH resulted in a marked deviance from normal follicular activity. The aberrations were probably caused by the interruption of pulsatile secretion of LH (but not FSH) leading to decreased but prolonged oestradiol secretion.
Lectin histochemistry was used to perform in situ characterization of the glycoconjugates present in boar testis and epididymis. Thirteen horseradish peroxidase- or digoxigenin-labelled lectins were used in samples obtained from healthy fertile boars. The acrosomes of the spermatids were stained intensely by lectins with affinity for galactose and N-acetyl-galactosamine residues, these being soybean, peanut and Ricinus communis agglutinins. Sertoli cells were stained selectively by Maackia ammurensis agglutinin. The lamina propria of seminiferous tubules showed the most intense staining with fucose-binding lectins. The Golgi area and the apical part of the principal cells of the epididymis were stained intensely with many lectins and their distribution was similar in the three zones of the epididymis. On the basis of lectin affinity, both testis and epididymis appear to have N- and O-linked glycoconjugates. Spermatozoa from different epididymal regions showed different expression of terminal galactose and N-acetyl-galactosamine. Sialic acid (specifically alpha2,3 neuraminic-5 acid) was probably incorporated into spermatozoa along the extratesticular ducts. These findings indicate that the development and maturation of boar spermatozoa are accompanied by changes in glycoconjugates. As some lectins stain cellular or extracellular compartments specifically, these lectins could be useful markers in histopathological evaluation of diseases of boar testis and epididymis.
Cellular retinol binding protein 1 (CRBP1) is the cytosolic carrier for retinol. It is expressed in many tissues, but the concentrations vary considerably. In Sertoli cells from immature rat testis, CRBP1 is highly expressed. The results of the present study show that regulation of CRBP1 expression at the protein level appears to be independent of regulation at the mRNA level. In Sertoli cells from prepubertal 19-day-old rats, CRBP1 mRNA is strongly induced for up to 72 h by the presence of serum factors. In contrast, treatment of the cells with cAMP analogues led to a rapid reduction in mRNA to quantities less than 5% of control values. However, the changes in CRBP1 mRNA did not lead to similar changes in the concentration of CRBP1 protein during 72 h of observation. Similarly, treatment of cells from 32- and 44-day-old rats with serum led to increased CRBP1 mRNA, whereas cAMP treatment resulted in a decrease in CRBP1 mRNA. Again, no changes were observed in the concentration of CRBP1 protein. Furthermore, co-incubation of Sertoli cells from 19-day-old rats with purified pachytene spermatocytes or round spermatids resulted in an increase in mRNA for CRBP1. However, comparable changes in CRBP1 protein concentrations were not observed. Neither cAMP nor serum changed the fraction of CRBP1 mRNA that was associated with polysomes. As a possible explanation for some of the results, pulse-chase experiments showed that the rate of CRBP1 degradation in cultured Sertoli cells is decreased by cAMP. It is proposed that these changes at the level of protein turnover contribute to the maintenance of stable concentrations of CRBP1 even when the corresponding mRNA concentrations vary markedly.
The role of LH in luteal function in pregnant dogs was investigated at two different periods during pregnancy: (i) the transitional period from apparent total independence of the corpus luteum to relative hormonal dependence (days 20-35); and (ii) the period of full hormonal dependence (days 35-40). At both periods, LH neutralization, LH inhibition and LH administration studies were conducted. At both periods LH immunoneutralization had no significant effect on the secretion pattern of progesterone or prolactin. GnRH antagonist treatment (Nal-Glu) decreased plasma LH below the detection limit in all treatment periods. Nal-Glu had no effect on prolactin. When GnRH antagonist osmotic pumps were implanted, a transient decrease in plasma progesterone concentrations occurred on days 21-22 but not during the remaining implantation period. When GnRH antagonist was injected, plasma progesterone temporarily decreased (24 h) after the beginning of treatment starting on day 20, but decreased for 5 days when the treatment started on day 35. When purified pig LH was injected i.v. twice a day for 2 consecutive days either from day 30 or from day 40, plasma progesterone concentrations remained constant during treatment. However, on days 40 and 41, an increase in prolactin was observed. These results indicate that LH immunoneutralization may not impair corpus luteum function. In addition, GnRH antagonist induces dose- and time-dependent effects. Only high doses resulted in a decrease in progesterone, the duration of which increased as pregnancy progressed. Continuous GnRH antagonist administration, even when associated with complete LH inhibition, was not associated with detectable effects on progesterone. Finally, LH administration does not stimulate progesterone but may modify prolactin in the last third of pregnancy. Other studies indicated a corpus luteum prolactin dependency. The present study indicates that, in pregnant bitches, LH may not be necessary to sustain progesterone synthesis but that its role may vary in a time-dependent manner.
The cellular mechanisms underlying the formation and organization of the human placenta remain poorly understood. Recent studies have demonstrated that E-cadherin, in association with the cytoplasmic protein known as beta-catenin, plays an integral role in the differentiation of the trophectoderm in the murine and bovine embryo. Although E-cadherin expression is regulated during the aggregation and fusion of human villous cytotrophoblasts, the expression of beta-catenin during the terminal differentiation of these primary cell cultures has not been determined. In this study, beta-catenin mRNA concentrations and protein expression were examined in primary cultures of human villous cytotrophoblasts using northern and western blot analysis. beta-catenin mRNA concentrations and protein expression were high in freshly isolated mononucleate cytotrophoblasts but decreased as these cells underwent aggregation and fusion to form syncytium. A similar pattern of expression was observed for the E-cadherin mRNA transcript and protein species present in these cell cultures. Immunoprecipitation studies demonstrated that the beta-catenin and E-cadherin protein species present in the mononucleate cytotrophoblasts were capable of forming intracellular complexes. In contrast, beta-catenin and E-cadherin mRNA and protein expression in JEG-3 choriocarcinoma cells remained constant over time in culture. beta-catenin and E-cadherin expression was subsequently immunolocalized to the aggregates of mononucleate cells present in both of these trophoblastic cell cultures and the villous cytotrophoblasts of the human first trimester and term placenta. Taken together, these observations indicate that the E-cadherin-beta-catenin complex plays a central role in the terminal differentiation of human trophoblasts in vitro and in vivo.
In the present study follicles were classified according to the morphology of their granulosa cells. Type B follicles contained only flattened granulosa cells; type B/C follicles had a mixture of flattened and cuboidal granulosa cells in a single layer, and type C follicles had a single layer of cuboidal granulosa cells. The primary objectives of the study were to determine whether 5-bromo-2-deoxyuridine incorporation into type B/C follicles was a marker for initiation of growth and how long type B/C follicles could remain at the same stage before transformation to type C follicles. Female Holtzman rats received bromo-deoxyuridine for 7 days. After the infusion (day minipumps were removed = day 0), rats were ovariectomized on days 0 (n = 9), 30 (n = 8), 90 (n = 8) and 150 (n = 9). The numbers of type B, B/C and C follicles within one ovary were determined using modified fractionator counting. Analysis over all times demonstrated that there were more (P < 0.0001) type B/C (941 +/- 61 per ovary) than type C (140 +/- 18 per ovary) or type B (159 +/- 19 per ovary) follicles. The numbers of type B and type C follicles did not differ from each other at any time. Only one of 34 rats evaluated had bromo-deoxyuridine-labelled type B follicles. On day 150, 57% of the bromo-deoxyuridine-labelled type B/C follicles remained from day 0. It is concluded that (1) DNA synthesis in granulosa cells of type B/C follicles is not a reliable indicator of impending growth; and (2) type B and type B/C follicles are both components of the pool of primordial follicles.
The aim of this study was to characterize the immediate effects of heat stress on plasma FSH and inhibin concentrations, and its involvement in follicular dynamics during a complete oestrous cycle, and to examine a possible delayed effect of heat stress on follicular development. Holstein dairy cows were oestrous synchronized and randomly assigned to either cooled (n = 7) or heat-stressed (n = 6) treatment groups. During a complete oestrous cycle, control cows, which were cooled, maintained normothermia, whereas heat-stressed cows, which were exposed to direct solar radiation, developed hyperthermia. At the end of this oestrous cycle (treated cycle), both groups were cooled and maintained normothermia for the first 10 days of the subsequent oestrous cycle. Throughout this period, follicular development was examined by ultrasonography, and plasma samples were collected. During the second follicular wave of the treated oestrous cycle, a significantly larger cohort of medium sized follicles (6-9 mm) was found in heat-stressed cows than in cooled cows (P < 0.05). The enhanced growth of follicles in this wave in heat-stressed cows was associated with a higher plasma FSH increase which lasted 4 more days (days 8-13 of the oestrous cycle; P < 0.05), and coincided with a decrease in the plasma concentration of immunoreactive inhibin (days 5-18 of the oestrous cycle; P < 0.05). During the follicular phase (days 17-20 of the treated cycle), heat-stressed cows showed an increase in the number of large follicles (>/= 10 mm), and the preovulatory plasma FSH surge was significantly higher in heat-stressed cows than in cooled cows (P < 0.01). The effect of heat stress was also observed during the first follicular wave of the subsequent cycle: the postovulatory plasma FSH concentration was higher (P < 0.01), but fewer medium follicles developed, and the first follicular wave decreased at a slower rate in previously heat-stressed cows than in cooled cows (0.40 and 0.71 follicles per day, respectively). This study shows both immediate and delayed effects of heat stress on follicular dynamics, which were associated with high FSH and low inhibin concentrations in plasma. These alterations may have physiological significance that could be associated with low fertility of cattle during the summer and autumn.
At birth, the mouse gonocyte does not resume mitotic activity for several days in vivo but, in an in vitro clonogenic system, cell division commences soon after culture. Somatic testis cell underlays had potent inhibitory activity on gonocyte-derived colony formation (23 +/- 15% compared with 84 +/- 1% in controls; P = 0.0001) when added to cultures of gonocytes in vitro. A Sertoli cell line, TM4B, had an even more pronounced effect on gonocyte clonogenic capacity, with 1 +/- 1% compared with 72 +/- 17% colony formation in controls (P = 0.0003). Testis cells appeared to have a direct inhibitory effect since testis-conditioned medium did not show a significant reduction in the number of colonies. The observed reduction in colony formation with the testis cell underlay was not accounted for by decreased attachment of gonocytes as simultaneous addition of a single cell suspension of testis cells was still effective in significantly reducing colony number when compared with controls (P = 0.01). Therefore, the observed inhibition exerted by testis cells appears to be a consequence of decreased proliferation of gonocytes. Growth factors belonging to the transforming growth factor beta superfamily which are known to be expressed in testis, such as transforming growth factor beta and epidermal growth factor, did not exert any inhibitory action on gonocyte-derived colony formation when added together or alone. However, a shift to a smaller colony size occurred in the presence of transforming growth factor beta and transforming growth factor beta plus epidermal growth factor, indicating a reduction in colony cell proliferation. Evidence for the expression of the Müllerian inhibiting substance receptor on newborn gonocytes using in situ hybridization was inconclusive. This finding was in agreement with the lack of a direct action of Müllerian inhibiting substance on the formation of gonocyte-derived colonies in vitro. Leukaemia inhibitory factor, alone or in combination with forskolin, had neither an inhibitory nor an enhancing effect on gonocyte-derived colony formation. An in vitro clonogenic method to assay for the proliferation of gonocytes in the presence of specific growth factors, cell lines, testis cell underlays and cell suspensions was used to identify a somatic cell-mediated inhibitor which may be responsible for the inhibitory action on gonocyte proliferation in vivo shortly after birth.
Despite many efforts, the control of reproduction in feral cat populations is still a problem in urban regions around the world. Immunocontraception is a promising approach; thus the present study examined the suitability of the widely used pig zona pellucida proteins (pZP) for contraception in feral domestic cats. Purified zona pellucida proteins obtained from pig and cat ovaries were used to produce highly specific antisera in rabbits. Antibodies against pZP raised in rabbits or lions were not effective inhibitors of either in vitro sperm binding (cat spermatozoa to cat oocytes) or in vitro fertilization in cats, whereas antibodies against feline zona pellucida proteins (fZP) raised in rabbits showed a dose-dependent inhibition of in vitro fertilization. Immunoelectrophoresis, ELISA and immunohistology of ovaries confirmed these results, showing crossreactivity of anti-fZP sera to fZP and to a lesser extent to pZP, but no interaction of anti-pZP sera with fZP. It is concluded that cat and pig zonae pellucidae express a very small number of shared antigenic determinants, making the use of pZP vaccine in cats questionable. A contraceptive vaccine based on feline zona pellucida determinants will be a better choice for the control of reproduction in feral cats if immunogenity can be achieved.
A considerable number of spermatozoa are used in each sow in routine artificial insemination. However, within a few hours after insemination, many spermatozoa are phagocytosed by polymorphonuclear leucocytes. Some aspects of sperm transport in the female genital tract in the sow have been thoroughly investigated, whereas little is known about the mechanisms involved in the phagocytosis of spermatozoa, or about which spermatozoa (fresh, capacitated or dead) are the most susceptible to ingestion by polymorphonuclear leucocytes. In this study, phagocytosis was investigated by use of an in vitro phagocytosis assay. Polymorphonuclear leucocytes were challenged with either untreated, cold-shocked or frozen-thawed spermatozoa, or with spermatozoa that had been treated to induce capacitation in vitro. The influence of serum on phagocytosis was also investigated. Treatment of the semen to induce capacitation in vitro considerably reduced the phagocytosis of spermatozoa, whereas crude treatments like cold-shock or freezing and thawing reduced phagocytosis only in the first 15-30 min of incubation with polymorphonuclear leucocytes. Viable spermatozoa were phagocytosed mainly through a pathway that was independent of complement or other serum components (for example, antibodies). Complement had little effect on phagocytosis of spermatozoa, but did cause acrosomal exocytosis and cell death.
Insulin-like growth factor I (IGF-I) is believed to play a luteotrophic role in the pig corpus luteum during the oestrous cycle. Since the actions of IGF-I in target tissues are mediated by the type I IGF receptor, the concentrations of IGF-I receptor mRNA and protein were examined in pig corpora lutea at different stages of the oestrous cycle. Corpora lutea were collected from normally cyclic gilts on days 4, 7, 10, 13, 15 and 16 of the oestrous cycle (n = 4 animals per day). Corpora lutea on days 7, 10 and 13 were dissociated with collagenase, and large and small luteal cell sub-populations were separated by elutriation. Northern and slot blots were used to examine mRNA, and western blots were used to measure the concentrations of IGF-I receptor protein in the pig corpus luteum. On northern blots, luteal IGF-I receptor mRNA was present as a single 11 kb transcript. The slot blots showed that the steady state expression of IGF-I receptor mRNA increased significantly (P < 0.05) from its lowest value on day 4, to reach a maximum on days 13-16. IGF-I receptor mRNA was also expressed to a greater extent in large compared with small luteal cells (P < 0.05). On western blots, IGF-I receptor appeared as a 95 kDa protein band (beta-subunit) and IGF-I receptor protein concentrations were significantly higher (P < 0.05) on days 4-10 than on days 13-16. Finally, large luteal cells appeared to contain more IGF-I receptor protein than the small luteal cells. In conclusion, since IGF-I receptor was detected in the pig corpus luteum, it is a likely target tissue for IGF-I, especially during the early luteal phase. Furthermore, IGF-I receptor was localized primarily on large luteal cells, thus it is hypothesized that IGF-I may play a paracrine role in the pig corpus luteum.
In mares, the shortage of oocytes and the variability in nuclear maturation at a certain time of the oestrous cycle hinders the optimization of methods for in vitro maturation and in vitro fertilization. Increasing the number of small-to-medium-sized follicles available for aspiration in vivo may increase the overall oocyte yield. The aims of the present study were to investigate whether administration of crude equine gonadotrophins affects follicular development, oocyte recovery rate, in vivo oocyte maturation and follicular concentrations of meiosis-activating sterols. During oestrus, all follicles >/= 4 mm were aspirated from 19 pony mares (first aspiration: A1). Over the next 8 days, the mares were treated daily with either 25 mg crude equine gonadotrophins (n = 10) or physiological saline (n = 9). Between day 1 and day 8, follicular growth was monitored by ultrasonography. On day 8, all follicles >/= 4 mm were evacuated (second aspiration: A2) and nuclear maturation of the recovered oocytes was assessed after orcein staining. Follicular growth between A1 and A2, as well as the number and size of follicles at A2 were similar for control mares and mares treated with crude equine gonadotrophins. The oocyte recovery rates at A1 and A2 were similar. At A2, the oocyte recovery rate and oocyte maturation in vivo were not affected by treatment with crude equine gonadotrophins. The number of expanded cumulus oophorus complexes recovered from follicles </= 29 mm was significantly higher at A1 than at A2. The number of oocytes at the germinal vesicle stage was significantly higher at A2 (41.5%) than at A1 (17.8%). Meiosis-activating sterols (FF-MAS and T-MAS) were identified in follicular fluid recovered at A2. Follicular concentrations of FF-MAS and T-MAS were unaffected by treatment with crude equine gonadotrophins. The present study demonstrates that follicular aspiration during oestrus allowed a new follicular population to develop and resulted in a higher degree of synchronization of oocyte development with respect to cumulus expansion and nuclear maturation. The availability of a more homogeneous population of oocytes might facilitate a better optimization of in vitro maturation and in vitro fertilization techniques in mares. Administration of crude equine gonadotrophins during early dioestrus did not affect the growth of small follicles, the oocyte yield after aspiration or oocyte maturation in vivo.
Sumatran rhinoceros (Dicerorhinus sumatrensis) sperm samples were collected from a post-copulatory female and characterized to determine their potential for sperm preservation and future use in artificial insemination. Five samples of acceptable quality from one male were used to compare the effect of two cryoprotectants (glycerol and dimethyl sulfoxide (DMSO)) and two post-thaw protocols (untreated and glass wool column) on sperm quality. The percentage of motile spermatozoa, sperm motility index (0-100) and sperm morphology were evaluated subjectively, and viability and acrosomal status were assessed using fluorescent markers. Evaluations of frozen-thawed spermatozoa were performed over a 6 h incubation interval. Post-coital semen samples (n = 5; 104.0 +/- 9.1 ml; 2.5 +/- 0.8 x 10(9) total spermatozoa; mean +/- SEM) exhibited a sperm motility index of 56.7 +/- 3.3, and contained 40.2 +/- 6.3%, 72.0 +/- 3.2% and 79.8 +/- 6.5% normal, viable and acrosome-intact spermatozoa, respectively. Glycerol and DMSO were equally effective as cryoprotectants and, regardless of post-thaw protocol, samples retained greater than 80% of all pre-freeze characteristic values. Processing semen samples through glass wool yielded higher quality samples, but only half the total number of motile spermatozoa compared with untreated samples. High values for pre-freeze sperm characteristics were also maintained after cryopreservation of epididymal spermatozoa from one black rhinoceros (Diceros bicornis) using the same protocol. In summary, Sumatran rhinoceros spermatozoa of moderate quality can be collected from post-copulatory females. Rhinoceros sperm samples show only slight reductions in quality after cryopreservation and thawing and have potential for use in artificial insemination.
Lutein cells produce progestins that support pregnancy. Steroidogenesis requires coordination of the anabolic and catabolic pathways of lipid metabolism. Peroxisome proliferator-activated receptors (PPAR) are transcription factors that are central in the regulation of lipid metabolism. Hence, they may play a role in regulation of the development and regression of the corpus luteum. The present study investigated the expression of PPAR-gamma, n during different stages of the corpus luteum. Lutein cells were isolated mechanically from non-pregnant and pregnant heifers on days 5, 12 and 20 of the oestrous cycle (n = 3 for each day). PPAR-gamma in single cells was analysed by flow cytometry. PPAR-gamma 1 and PPAR-gamma 2 isoforms were distinguished by immunoblotting. The cell cycle of the lutein cells was measured by the flow cytometric quantification of DNA in single cells, using propidium iodide staining after ethanol fixation and RNAse treatment, and by the detection of the proliferating cell nuclear antigen (PCNA). The response of the cells to PPAR-gamma agonist 15-deoxy-delta 12,14 prostaglandin J2 (15dPGJ2, 200 and 490 nmol l-1) with and without changing the cell cycle by the anti-apoptogenic drug aurintricarboxylic acid (ATA, 10 mumol l-1) was used as an in vitro model to study the relationship between the cell cycle and PPAR-gamma. The concentration of PPAR-gamma per cell from non-pregnant heifers was significantly higher on day 5 (3.40 +/- 0.30 fmol) compared with that on day 12 (1.34 +/- 0.18 fmol, P < 0.05) and day 20 (0.55 +/- 0.2 fmol, P < 0.05). In pregnant heifers, the concentration of PPAR-gamma was significantly (P < 0.01) higher than in non-pregnant heifers. A decrease in the PPAR-gamma 1 isoform relative to PPAR-gamma 2 was observed in cells on day 12 of the oestrous cycle compared with day 5. The cell cycle (S phase portion in cells on days 5, 12 and 20: 16 +/- 4%, 6 +/- 4% and 4 +/- 3%, respectively) and the portion of cells with PCNA correlated with the amount of PPAR-gamma in non-pregnant heifers. ATA promoted the S phase in cells of non-pregnant heifers (day 12) and the endogenous agonist of PPAR-gamma, 15dPGJ2, inhibited the response to ATA in a dose-dependent manner, indicating that PPAR-gamma plays a role in the arrest of the cell cycle in lutein cells to maintain their differentiated state.
Endocrine control of follicular growth was studied in mature Romanov ewes carrying (RF+) or not carrying (R+2) the Booroola Fec gene during an oestrous cycle after gonadotrophin-dependent follicles were suppressed by treatment with an antagonist of GnRH (Antarelix, 0.5 mg per day) and superovulatory treatment was administered. The left ovary was removed after 10 days of treatment (saline or Antarelix) and the right ovary was removed at the end of the superovulatory treatment. Ewes of both genotypes treated with Antarelix had lower plasma LH concentrations than did controls from day 0 to day 10. The inhibitory effect of Antarelix on LH concentration increased with day of treatment. The variability in FSH concentrations during the initial 10 days was reduced by Antarelix treatment in both genotypes. Plasma FSH concentrations were higher in RF+ ewes than in R+2 ewes. In both genotypes, FSH concentrations varied significantly with day of treatment, with the lowest concentrations at day 8 and the highest concentrations at day 5. RF+ ewes had a greater total and atretic number of antral follicles 0.62-1.12, 1.12-2.00 and 2.00-3.00 mm in diameter (classes 2, 3 and 4) than did R+2 ewes before and after superovulatory treatment. After superovulatory treatment, the total number of atretic and non-atretic follicles > 3.00 mm in diameter (class 5) increased in both genotypes. Superovulatory treatment also increased the number of total and atretic class 4 follicles in RF+ only. Conversely, superovulatory treatment decreased the mean number of class 3 follicles in both genotypes, while the number of atretic follicles was decreased only in R+2 ewes. Antarelix treatment significantly reduced the percentage of follicles > 2.00 mm in diameter in RF+ but not in R+2 ewes. Antarelix treatment before superovulatory treatment increased the total number of class 4 follicles in both genotypes but the increase was more significant in RF+ than in R+2 ewes. These results indicate that Antarelix pretreatment favours a greater superovulatory response in Romanov ewes carrying the Fec gene because ovulatory follicles are recruited from a wider range of follicular size classes.
Prostaglandin F(2alpha) (PGF(2alpha)) is implicated in the process of luteal regression in many species. Treatment of rat luteal tissue with PGF(2alpha) increases the generation of reactive oxygen species. Since reactive oxygen species have been implicated in apoptosis, the present study was undertaken to determine whether reactive oxygen species play a role in the PGF(2alpha)-induced apoptosis of rat luteal cells. Rat luteal cells were loaded with 6-carboxy-2, 7'-dichlorodihydro-fluorescein (CDCFH) diacetate, di (acetomethyl ester), which can be oxidized by reactive oxygen species to yield CDCF, a fluorescent molecule, and the cells were treated with different doses of PGF(2alpha). Incubation with 100 micromol PGF(2alpha) l(-1) induced an increase in CDCF fluorescence (P < 0. 05). Treatment of cells with PGF(2alpha) for 48 h in serum-free medium induced a dose-dependent increase in cell death, and these cells exhibited the morphological characteristics typical of apoptosis, including condensed or fragmented nuclei and fragmentation of internucleosomal DNA. Pretreatment of these cells with ascorbic acid, N,N'-dimethylthiourea, or superoxide dismutase, which acts as an antioxidant or a radical scavenger, prevented the PGF(2alpha)-induced apoptosis. These results demonstrate that PGF(2alpha) produces reactive oxygen species and induces apoptosis in rat luteal cells, indicating that the reactive oxygen species may induce apoptotic cell death during luteolysis.
Interleukin 5 is expressed in type 2 T lymphocytes and has a key role in driving the differentiation, recruitment and activation of eosinophils. Mice with a null mutation in the interleukin 5 gene (IL-5 -/- mice) have altered type 2 immune responses and severely depleted eosinophil populations. In the present study, the effect of interleukin 5 deficiency on the abundant population of eosinophils present in the female reproductive tract was investigated, and the reproductive performance in C57Bl/6 IL-5 -/- mice was measured. Endometrial eosinophils, detected on the basis of their endogenous peroxidase activity, were reduced in number by four-sevenfold during the oestrous cycle and in early pregnancy in IL-5 -/- mice. Eosinophils present in the cervix and decidual tissues at the time of parturition were similarly diminished. The temporal fluctuations in eosinophil recruitment and localization within these tissues were otherwise unchanged, indicating that interleukin 5 is not a necessary chemotactic agent in the female reproductive tract. Oestrous cycles were moderately greater in duration in IL-5 -/- mice (mean +/- SD = 5.6 +/- 1.0 days in IL-5 -/- mice versus 5.0 +/- 0.8 days in IL-5 +/+ mice), owing to an extended period in oestrus (2.7 +/- 0.9 days per cycle in IL-5 -/- mice versus 1.8 +/- 0.7 in IL-5 +/+ mice). The interval between placing females with males and the finding of copulatory plugs was reduced significantly in interleukin 5-deficient mice. Implantation rates and subsequent fetal development were comparable in IL-5 -/- and IL-5 +/+ mice, irrespective of whether pregnancies were sired by syngeneic (C57Bl/6) or allogeneic (CBA or Balb/c) males, apart from a 10% increase in placental size and a 6.5% decrease in placental∶fetal ratio seen on day 17 in pregnancies sired by CBA males. Parturition and post-partum uterine repair were not compromised in interleukin 5-deficient mice, as judged by the length of gestation, and the outcomes of pregnancies initiated at post-partum oestrus. The birth weights and growth trajectories of pups were significantly influenced by interleukin 5 status, with small but significant increases in the weights of IL-5 -/- pups, particularly C57Bl/6 and CBA F(1) animals, remaining evident until adulthood. These data are consistent with the view that eosinophils have a role in endometrial tissue remodelling associated with the oestrous cycle, but indicate that the events of pregnancy and parturition proceed quite normally in the absence of maternal and fetal interleukin 5. However, strain-dependent effects of interleukin 5 deficiency on placental growth and function and subsequent weight gain in the newborn indicate that this cytokine may act through the maternal or fetal immune axis to exert subtle influences on reproductive outcome.
Changes in food intake affect the reproductive axis in both sexes, and the nutritional signals involved and the sites that receive those signals are now beginning to be unravelled. Our studies have focussed on the mature male sheep, a model in which high food intake stimulates GnRH-LH pulse frequency for only 10-20 days but continues to promote testicular growth over several months. Different signals and different target organs seem to be responsible for these short- and long-term responses. Short-term dietary treatments lead to changes in blood concentrations of glucose, fatty acids, insulin and leptin, and concentrations of glucose, insulin, leptin and some amino acids in cerebrospinal fluid. It seems unlikely that amino acids affect GnRH-LH secretion directly in sheep. Intracerebroventricular infusions of insulin specifically increase LH pulse frequency, but intravenous, intra-abomasal or intracerebroventricular infusions of glucose have no effect, despite their effects on cerebrospinal fluid insulin concentrations. The addition of fatty acids to the diet also increases LH pulse frequency, but does not affect the concentrations of insulin or leptin in the cerebrospinal fluid. It appears that acute responses to changes in nutrition involve a range of alternative pathways, possibly including interactions among insulin, leptin and energy substrates. Effects of long-term dietary treatments on testicular size are only partly dependent on the GnRH-LH system (that is, on brain control) and so must also depend on other, as yet unknown pathways. Concepts of 'metabolic sensing and integration' are being developed from the basis of existing knowledge of the central control of appetite and reproduction.
The spermatozoa of the musk shrew, Suncus murinus, have a fan-like giant acrosome with a diameter of approximately 20 mm. The aim of this study was to investigate how this giant acrosome is constructed in the musk shrew spermatid and, in particular, how the Golgi apparatus involved in acrosome formation behaves. The behaviour of the Golgi apparatus was monitored by confocal laser scanning microscopy with antibody against a Golgi-associated Rab6 small GTPase. In the early Golgi phase, small Golgi units, the Golgi satellites, localized as a large aggregate in the juxtanuclear cytoplasm. As acrosome formation progressed, the Golgi satellites gradually dispersed, associated with proacrosomal vesicles and an acrosomal vesicle, and finally became distributed as multiple small units over the whole surface of an acrosomal cap in the round spermatid. The mode of acrosome formation in musk shrews was distinctly different from that in rats and mice, in which the Golgi apparatus remains as a single unit throughout acrosome formation. In musk shrews, the proacrosomal vesicles formed successively by the Golgi satellites coalesced, one after another, into a potential acrosomal vesicle. This process may result in further enlargement of the acrosome. The results of the present study indicate that Golgi satellites are necessary for the biogenesis and development of the giant acrosome in musk shrew spermatozoa.
