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Advanced reproductive technologies are being applied for the propagation of squirrel monkeys, to ensure their preservation as a genetic resource and the effective use of their gametes in the future. In the present study, oocytes and spermatozoa were collected from live squirrel monkeys, following which piezo intracytoplasmic sperm injection (ICSI) was performed using these gametes. Follicular development was induced by administering equine chorionic gonadotropin (eCG) containing inhibin antiserum to an immature squirrel monkey female. The unilateral ovary was excised after the administration of human chorionic gonadotropin (hCG), to induce ovulation, following which the larger developed follicular oocytes were collected. Follicular oocytes were prepared for ICSI using sperm from the epididymal tail of a unilateral testis extracted from a mature male. The embryos were continuously incubated in CMRL 1066 medium supplemented with 10% (v/v) fetal bovine serum. Embryo culture was performed with cumulus cells. Two experiments of ICSI carried out with three females resulted in 14 mature oocytes from the 49 cumulus-oocyte complexes collected and five embryos, three of which developed into blastocysts. These blastocysts were vitrified, thawed, and transferred to recipient monkeys, but no pregnancies resulted. In conclusion, the present study is the first to successfully produce ICSI-derived blastocysts from MII oocytes obtained by means of hormone administration (a combination of eCG+inhibin antiserum and hCG) and in vitro maturation in immature squirrel monkeys.

The Ski protein is implicated in the proliferation/differentiation of a variety of cells. We previously reported that the Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. However, granulosa cells cannot only undergo apoptosis but can alternatively differentiate into luteal cells. It is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to determine the localization of the Ski protein in the rat ovary during luteinization to examine if Ski might play a role in this process. In order to examine the Ski protein expression during the progression of luteinization, follicular growth was induced in immature female rats by administration of equine chorionic gonadotropin, and luteinization was induced by human chorionic gonadotropin treatment to mimic the luteinizing hormone (LH) surge. While no Ski-positive granulosa cells were present in the preovulatory follicle, Ski protein expression was induced in response to the LH surge and was maintained after formation of the corpus luteum (CL). Although the Ski protein is absent from the granulosa cells of the preovulatory follicle, its mRNA (c-ski) was expressed, and the level of c-ski mRNA was unchanged even after the LH surge. The combined results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggested that its expression is regulated posttranscriptionally.

The present study was conducted to evaluate the endocrinological effects of the pituitary on luteal maintenance and regression in the cyclic golden hamster (Mesocritus auratus). After hypophysectomy (Hypox) at 0900 h on day 1 of the estrous cycle (the day of ovulation), the animals received injection of prolactin (PRL) or PRL plus equine chorionic gonadotropin (eCG). They were decapitated at 1500 h on day 3 of the cycle, and trunk blood was collected for measurement of progesterone (P4). Corpora lutea (CLs) were dissected from one ovary for DNA ladder detection by electrophoresis, determination of DNA fragmentation ratio by fluorometric measurement method and measurement of P4. The other ovary was used for histological observation. After the Hypox, the daily injection of 1 mg ovine PRL restrained the DNA fragmentation ratio and number of apoptotic cell in the CLs. The PRL treatment maintained the luteal morphology and increased the luteal P4 concentration, but not in the plasma P4 concentration. In addition to PRL, injection of 2 IU eCG after the Hypox also restrained the DNA fragmentation ratio and number of apoptotic cells in the CLs to the level of a pregnant animal. The PRL plus eCG treatment maintained the luteal morphology in the same manner as the PRL only treatment and increased not only the luteal but also the plasma P4 concentration. These results suggest that PRL restrains luteal apoptosis and maintains luteal morphology and that the combination of PRL and eCG restrains not only structural but also functional luteal regression in the cyclic hamster.

Cyclic AMP (cAMP) is a second messenger that plays a critical role in follicular recruitment, development and luteinization in the mammalian ovary. The cellular level of cAMP is largely dependent on the activity of phosphodiesterase (PDE), which degrades cAMP into 5'-AMP. The present study was conducted to investigate the level of cAMP and the activity of cAMP-PDE in postnatal rats; immature rats during gonadotropin-primed follicular development, ovulation and luteinization; adult rats during normal estrous cycling; and aged rats that spontaneously developed persistent estrous (PE) by radioimmunoassay (RIA). All four rat models were confirmed by histological examination of one ovary and assayed using the other ovary by RIA. In the postnatal rats, the ovarian cAMP level was high on day 10 after birth, while ovarian cAMP-PDE activity was highest at 21 days of age. In the immature female rats, both the ovarian cAMP level and cAMP-PDE activity increased remarkably after treatment with equine chorionic gonadotropin (eCG), increased continuously 24 h after injection of human chorionic gonadotropin (hCG) for induction of ovulation and luteinization, and then declined significantly. In the adult rats during the normal estrous cycle, the ovarian cAMP levels were low on the day of estrus, and there were no significant changes in ovarian cAMP-PDE activity throughout the estrous cycle. In the PE rats, the ovarian cAMP levels were similar to those of the adult rats on the day of estrus but were lower than those on the other days of the estrous cycle; ovarian cAMP-PDE activity was lower than that in the adult rats on any day of the estrous cycle. Together, these findings indicate that the ovarian cAMP level and cAMP-PDE activity were regulated in a stage-dependent manner during ovarian follicular development, atresia and luteinization and providing evidences that cAMP and cAMP-specific PDEs are involved in these physiological processes.

The aim of the present study was to locate Ski protein, a product of cellular protooncogene c-ski, in rat ovaries in order to predict the possible involvement of Ski in follicular development and atresia. First, expression of c-ski mRNA in the ovaries of adult female rats was confirmed by RT-PCR. Then, ovaries obtained on the day of estrus were subjected to immunohistochemical analysis for Ski and proliferating cell nuclear antigen (PCNA) in combination with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). Ski was expressed in granulosa cells that were positive for TUNEL, but negative for PCNA, regardless of the size of follicles. Expression of Ski in TUNEL-positive granulosa cells, but not in PCNA-positive granulosa cells, was also verified in immature hypophysectomized rats having a single generation of developing and atretic follicles by treatment with equine chorionic gonadotropin. These results indicate that Ski is profoundly expressed in the granulosa cells of atretic follicles, but not in growing follicles, and suggests that Ski plays a role in apoptosis of granulosa cells during follicular atresia.

The aim of the present study was to assess follicular and oocyte development in the same gilts during three phases of their reproductive life [prepuberal gilts (PP; 6.0 months of age), puberal gilts (P; 9.5 months of age) and primiparous sows (S)]. Follicular development was stimulated by the injection of 1,000 IU of equine chorionic gonadotropin (eCG) followed by 500 IU of human chorionic gonadotropin (hCG) 72 h later. Cumulus-oocyte-complexes (COCs) were recovered by endoscopic ovum pick up/aspiration from preovulatory follicles of the left ovary, and the follicular fluid (FF) from the right ovary was collected 34 h after the hCG treatment by endoscopy. Altogether, 19 pigs were used in the PP and P trials and 12 in the S trial. From the left ovaries, 168, 190 and 82 follicles were aspirated and 106, 125 and 42 COCs, respectively, were recovered (recovery rate 61 +/- 27, 63 +/- 21 and 53 +/- 22%, respectively). The mean number of follicles was greater in the P phase than in the PP phase (19.7 +/- 6.8 vs. 15.7 +/- 6.8; p=0.06) and S phases (14.2 +/- 4.0; p

The effects of passive immunoneutralization of endogenous inhibin on ovulation rate in immature rats were investigated. Efficiency of superovulation on production of fertilized oocytes was compared between the inhibin antiserum (inhibin-AS) and equine chorionic gonadotropin (eCG) protocols. Immature female Wistar strain rats were superovulated with a single injection of 100-200 microl inhibin-AS, with and without an injection of human chorionic gonadotropin (hCG). A total of 77.8% of the 26-30-day-old rats treated with a single injection of 100-200 microl inhibin-AS ovulated 72 h after treatment, while rats given normal goat serum (NGS; 200 microl) did not ovulate. At 28 days of age, all of the inhibin-AS treated rats ovulated when additional hCG treatment was given, whereas the number of ovulated oocytes was not affected. The number of ovulated oocytes in the inhibin-AS-hCG treated groups was significantly higher than that of the NGS-hCG treated group. In addition, plasma concentrations of FSH in the inhibin-AS-hCG treated group significantly increased compared with the NGS treated group. While the percentage of mated rats in the 200 microl inhibin-AS-hCG treated group was significantly lower than that of the 15 IU eCG-hCG treated group, the fertilization rate was comparable between the two groups. The number of fertilized oocytes in the 200 microl inhibin-AS-hCG treated group was significantly higher in comparison with the 15 IU eCG-hCG treated group. These results suggest that immunoneutralization of endogenous inhibin could be a reliable method for induction of superovulation to collect a large number of normally fertilized oocytes in immature rats.

The present study was conducted to investigate fertility of estrus-induced ewes during the non-breeding season, artificially inseminated with frozen semen imported from New Zealand. A total of 122 Suffolk and Suffolk-crossed ewes in three different sheep farms (T, S, M) were treated with an intravaginal progesterone release device (CIDR-G) for 12 days, and an intramuscular injection of 500 IU equine chorionic gonadotropin one day before CIDR removal. Effects of intrauterine insemination doses (0.2 and 0.4 ml per head) and two rams on fertility were compared at Farm T using 75 ewes. At the other two farms (S and M), 0.1 to 0.2 ml doses were inseminated into the uteri on the fixed-time basis. The lambing rates of ewes inseminated with 0.2 and 0.4 ml doses were 68.4 and 59.5%, and the lambing rates of ewes inseminated with the two rams were 61.3 and 65.9%. No significant differences were found between any of the factors. The lambing rates on Farms S and M were 54.2% and 47.8%, respectively. Overall, there was no significant difference in the lambing rates (58.3% and 59.5%) and prolificacy (2.03 and 1.61) between the two rams. These results indicate that the fertility of ewes inseminated during the non-breeding season with frozen-thawed semen imported from New Zealand is acceptable and that new blood lines of Suffolk sires and dams can be produced on Japanese sheep farms.

The present study was conducted to determine the effect of two human chorionic gonadotropin (hCG) treatments on the fertility of ewes which were estrus-induced and artificially inseminated during the non-breeding season. Suffolk (SF: n=68) and South-Down (SD: n=20) ewes were pretreated with a controlled internal drug release dispenser (CIDR) for 12 days and 500 IU equine chorionic gonadotropin 1 day before CIDR removal. Each breed of ewes were divided into 3 groups: Group I (n=31), an intramuscular injection of 100 IU hCG was given on Days 3, 4 and 5 after artificial insemination (AI) (Day 0=the day of AI); Group II (n=29), an injection of 300 IU hCG was given on Day 4; and Group III (n=28, Control), an injection of 0.6% saline was given on Day 4. Plasma progesterone (P4) concentrations were measured by enzyme-immunoassay in all ewes in the three groups at the time of insemination (Day 0), and Days 4, 8, 12, 16, and 20 after AI. On Day 8, the mean P4 concentrations in ewes treated with hCG (Groups I and II) were significantly higher (P

To clarify the endocrinological characteristics of bovine gonadal hypoplasia (XY female), peripheral FSH, LH, inhibin, estradiol-17β (E2), progesterone (P) and testosterone (T) levels were measured before and after the administration of eCG, hCG and GnRH-A in the 3 bovine XY females without the Sry gene. Before the administration of hormonal drugs, the concentrations of FSH and LH were higher than in adult bulls, cows in the luteal phase and castrated bulls. On the other hand, the concentrations of inhibin and E2 were lower than those observed in the follicular phase during the estrous cycle. Concentrations of P and T were much lower than those observed in the luteal phase during the estrous cycle and in bulls. Gonads of our XY females did not respond to eCG or hCG stimulation, whereas the pituitary was highly responsive to GnRH-A in LH secretion.

Two trials were conducted to investigate a simple superovulation method for Suffolk ewes during the breeding season. The method involves a single injection of porcine follicle-stimulating hormone (pFSH) combined with equine chorionic gonadotropin (eCG) and was evaluated by measuring the concentrations of plasma hormones (estradiol-17β(E2), progesterone (P4), luteinizing hormone (LH) and follicle-stimulating hormone (FSH)). The ewes were treated with a vaginal sponge containing synthetic steroid with progestative action (P sponge) for 12 days in both trials. In Trial 1 (conducted in 1995), 18 ewes received a single injection (F1) or multiple injections (F6) of a total of 20 mg pFSH, and in combination with an injection of 500 IU eCG (F1+P, F6+P). The injections were conducted on Day -2 (Day 0 was the day of P sponge removal). In trial 2 (conducted in 1996), 24 ewes were given single injections of pFSH and eCG at different times; (i) both hormones on Day -2 (F-2/P-2), (ii) pFSH on Day -2 and eCG on Day -1 (F-2/P-1), and (iii) both hormones on Day -1 (F-1/P-1). One hundred micrograms of an analogue of gonadotropin-releasing hormone (GnRH) was administered on Day 1 in Trial 1 and at the detection of estrus in Trial 2. Frozen-thawed semen was inseminated in utero using a laparoscope at 30 h and 38 h after P sponge removal in Trial 1 and at 18 hours after the detection of estrus in Trial 2. Blood samples were collected from the jugular vein. E2, P4, LH and FSH were assayed by second-antibody enzyme immunoassays. F1+P showed similar endocrine profiles to those of F6 and F6+P with a preovulatory E2 peak after P sponge removal, but F1 did not show a clear preovulatory E2 peak. The E2 concentration in F-2/P-1 rose biphasically; there was a rise just after injection of pFSH and then another rise after P sponge removal. Simultaneous injections of 20 mg pFSH and 500 IU eCG may reduce the E2 elevation. We concluded that a good ovarian response comparable to the multiple injection method can be achieved by the present simple superovulation method.

The incidence of premature corpora lutea (CL) regression was investigated in three different breeds (Merino × polled Dorset: MD, Suffolk: S, and South-Down: SD), parous and non-parous ewes (a total of 46 ewes) treated for superovulation during and out of the breeding season. Superovulation was induced with a single injection of 20 mg porcine follicle-stimulating hormone (pFSH) 2 days and 500 IU equine chorionic gonadotropin (eCG) 1 day, respectively before the removal of synthetic progestogen (40 mg FGA) vaginal sponges inserted for 12 days. All ewes were inseminated into the uterus with fresh-diluted or frozen-thawed semen by laparoscope, and a number of newly formed and regressed CL were recorded. Breed difference significantly (P

A model of induced follicular atresia consists of hypophysectomizing hamsters on the morning of estrus (day 1 of cycle) followed by injecting 30 IU equine chorionic gonadotropin (eCG). On the morning of day 4 (0 h) atresia is induced by administration of an antiserum to eCG (eCG-AS). One hour after eCG-AS, plasma estradiol and testosterone decline to 15.8% and 25.9%, respectively, of 0 h values with progressive decreases until at 12 h after eCG-AS, circulating estradiol is no longer detectable although baseline levels of testosterone (10.7%) are still present. The question raised was whether the simultaneous drop in both steroids at 1 h post eCG-AS is attributable to the fall in both hormones or to a more drastic decline in thecal androgens, thus depriving the granulosa of the precursors needed to synthesize estrogens. To answer this question, antral follicles were dissected 2 and 4 h after eCG-AS and incubated for 5 h, with or without the addition of 10 ng androstenedione. Addition of androgen at either time led to an increase in estradiol accumulation in the medium after the first hour which was sustained over the next 4 h. These experiments indicate that the immediate effect of eCG-AS is to sharply reduce thecal androgen production thus depriving granulosal aromatase of the necessary steroid substrate. In previous histological studies, we have shown that the earliest time when morphological signs of atresia are first discernable is by 4 h post eCG-AS as judged by pyknotic granulosa nuclei in the cumulus oophorus. In this study, using gel electrophoresis and autoradiography of 3’ labeled DNA followed by quantitating low and high molecular weight DNA, the earliest consistent onset of atresia was also at 4 h after eCG-AS.

The present study was undertaken to evaluate ovulation time after superovulation treatment in ewes during the non-breeding season. Ewes (n=17) were treated with vaginal sponges impregnated with fluorogesterone acetate (FGA sponge) for 12 days. To the superovulation group (n=9), an intramuscular injection of 20 mg porcine follicle stimulating hormone (pFSH) was given at 48 h before sponge removal, and 500 IU equine chorionic gonadotropin (eCG) was given 24 h later after the pFSH injection. Ewes in the control group (n=8), were given 500 IU eCG alone 24 h before sponge removal. The time of ovulation was assessed by repeated observation of the ovaries in both groups of ewes using a laparoscope. Observations were commenced 24 h after sponge removal and were conducted every 6 h for a total of 60 h. The intervals between sponge removal and the onset of estrus, and ovulations were shorter in the superovulation group than the control group (P

Two trials were conducted to investigate the effects of a simple superovulation method using a single injection of porcine follicle-stimulating hormone (pFSH) combined with equine chorionic gonadotropin (eCG) on ovarian responses and embryo qualities in Suffolk ewes during the breeding season. The ewes were treated with a vaginal sponge containing synthetic progestative steroid (P sponge) for 12 days in both trials. In Trial 1 (conducted in 1995), 18 ewes received a single injection (F1) or multiple injections (F6) of a total of 20 mg pFSH, and in combination with an injection of 500 IU eCG (F1+P, F6+P). The injections were conducted on Day -2 (Day 0, was the day of P sponge removal). In trial 2 (conducted in 1996), 24 ewes were given single injections of pFSH and eCG at different times: (i) both hormones on Day -2 (F-2/P-2), (ii) pFSH on Day -2 and eCG on Day -1 (F-2/P-1), and (iii) both hormones on Day -1 (F-1/P-1). One hundred micrograms of an analogue of gonadotropin-releasing hormone (GnRH) was administered on Day 1 in Trial 1 and at the detection of estrus in Trial 2. Frozen-thawed semen was inseminated in utero using a laparoscope at 30 h and 38 h after P sponge removal in Trial 1 and at 18 h after the detection of estrus in Trial 2. Embryos were recovered on Day 7 or Day 8. In Trial 1, the mean number of ovulations and the rate of normal embryos were 6.8, 16.7% for F1, 15.0, 36.0% for F1+P, 15.5, 27.0% for F6 and 20.3, 44.2% for F6+P. The F1+P group showed the results comparable to those of F6 and F6+P. In Trial 2, the mean number of ovulations were 17.8 for F-2/P-1, 13.1 for F-2/P-2 and 12.4 for F-1/P-1. The rate of normal embryos in F-2/P-1 (63.0%) was significantly higher than in F-2/P-2 (41.1%, P

The expression of inhibin α-subunit mRNA was examined in the corpus luteum (CL) of pregnant mares. Corpora lutea were obtained from pregnant mares at 50 h, 94 h after ovulation and on 30, 60, and 90 days of pregnancy. Northern blot analysis, using a cDNA specific to equine inhibin α-subunit as a probe revealed that the expression level of its transcript continued to decline until 30 days after ovulation. However, on day 60 of pregnancy, the level increased similar to that at 94 h after ovulation. On day 90 of pregnancy, the level declined again. The expression of inhibin α-subunit mRNA in the estrous cycle CL (7 days after ovulation) was also examined but the level was moderately low. Considering that serum concentration of equine chorionic gonadotropin (eCG) peaks at around day 60 of pregnancy, and the expression level of inhibin α-subunit in the CL of the pregnant mare also shows a peak on day 60 of pregnancy, it was suggested that, during pregnancy in equidae, the expression of inhibin α-subunit mRNA in the CL is stimulated by eCG.

In the present study, we investigated changes in plasma concentrations of inhibin, estradiol-17β, progesterone and FSH in the golden hamster (Mesocricetus auratus) superovulated by equine chorionic gonadotropin (eCG) treatment. In addition, the hamsters were injected at various times with human chorionic gonadotropin (hCG) to determine the follicular development after treatment with eCG. After treatment with 30 IU eCG at 1100 h on day 1 of the estrous cycle (day 1=day of ovulation), superovulation (36.5 ± 1.1 v.s. 13.5 ± 0.4 in controls treated with saline) occurred on the expected next day 1. Plasma concentrations of inhibin and estradiol-17β increased and plasma concentrations of FSH decreased in the animals given 30 IU eCG. Plasma concentrations of progesterone also increased after eCG treatment; however, they decreased to control levels within 48 h after eCG injection. The number of follicles capable of ovulating after hCG administration was dramatically increased within 36 h after eCG injection (43.4 ± 3.4). Thereafter, the number of follicles capable of ovulating decreased to about a half of the peak value during the next 6 h period, then increased gradually to the numbers of ovulations observed on the next day 1. Plasma inhibin and estradiol-17β were augmented and plasma concentrations of FSH were suppressed in the animals in which follicular development was stimulated, though there is a difference in the changing pattern between plasma inhibin and estradiol-17β. The present results also suggest that plasma concentrations of inhibin may be an indicator for the number of developing follicles and estradiol-17β may be an indicator for follicular maturation.

The process of spermatogenesis was studied in mutant hypogonadal (hpg) male mice treated with equine chorionic gonadotropin (eCG) or gonadotropin releasing hormone (GnRH) for 60 days. Untreated adult hpg male mice had very small gonads (5.7 ± 0.6, n=7, mean ± SEM) and no mature spermatozoa due to a genetic deficiency of GnRH. Testicular weights increased to 55.7 ± 5.6 mg in eCG (5 IU/day) injected mice (n=7) and 30.0 ± 3.8 mg in GnRH (1 μg/day) injected mice after 60 days. Similar increases were seen in seminal vesicles. In eCG-administered mice, matured spermatozoa appeared in epididymides after 45 days of treatment. A lower dose of eCG (2.5 IU/day) or GnRH (1 μg/day) were less effective for stimulation of spermatogenesis. Although the propotion of hpg mouse sperm fertilized embryos that developed to the blastocyst stage (18%) was lower than the proportion of normal mouse sperm fertilized embryos (43%), at least some of the hpg mouse sperm fertilized embryos produced viable offspring through embryo transfer. The present study proved that chronic eCG or GnRH administration could stimulate spermatogenesis in hpg males, and cauda epididymal spermatozoa were fertile when obtained from eCG-injected mutant mice. These results intimated that hpg male mice provide valuable information for clarifying the endocrine and paracrine control processes of spermatogenesis.

Equine chorionic gonadotropin (eCG) is a member of the glycoprotein family, which includes lutropin (LH), follitropin (FSH), and thyrotropin (TSH), all consisting of two noncovalently linked a-and β-subunits. In the present study, we determined the full length of the equine α-subunit cDNA sequence. mRNA was extracted from equine placenta on day 70 of gestation. The nucleotide sequence of the eCG a-subunit cDNA was amplified by PCR using mixed primers designed on the basis of the nucleotide sequence of human, rat and mouse a-subunits. Analysis of the nucleotide sequence revealed that the a-subunit amplified contained 387 bp which included the signal peptide (24 amino acids containing 4 amino acids expected) and the first 3 amino acid residues. When the expression of both subunit mRNAs in the equine placenta was compared by Northern blotting, the expression of the β-subunit mRNA was relatively greater than that of the a-subunit.