Regulation of Sertoli cell transferrin and sulfated glycoprotein-2 messenger ribonucleic acid levels during the restoration of spermatogenesis in the adult hypophysectomized rat.

The purpose of this study was to determine whether the levels of sulfated glycoprotein-2 (SGP-2) and/or transferrin mRNA in Sertoli cells responds to testosterone in the adult rat testis and, if so, to distinguish between the effects of testosterone and those of changing populations of germ cells. To this end we have examined changes in the steady state levels of these two mRNAs in relationship to changes in intratesticular testosterone concentration and germ cell numbers after treatment of adult hypophysectomized rats with testosterone. Hypophysectomy for 4 weeks caused intratesticular testosterone concentrations to become reduced from 50 to 3 ng/ml and caused significant reductions in the numbers of testicular spermatozoa (undetectable), round spermatids (nearly undetectable), and pachytene spermatocytes (12% of normal). Intratesticular testosterone concentrations rose to 30 ng/ml within 3 days after implantation of testosterone-containing polydimethylsiloxane capsules into the hypophysectomized rats. Three days after the initiation of testosterone treatment, increases were seen in the numbers of round spermatids (10% of normal) and pachytene spermatocytes (29% of normal). The major increases in the numbers of these cells and of spermatozoa occurred between days 14-56 of testosterone treatment, with pachytene spermatocytes reaching a maximum of 75% of the control level by day 28, and round spermatids and spermatozoa reaching 75% and 21% of the control levels, respectively, by 56 days. The steady state levels of SGP-2 mRNA were not affected by hypophysectomy, testosterone administration, or subsequent increases in germ cell numbers. In contrast, transferrin mRNA levels were reduced by hypophysectomy. As found for SGP-2, transferrin mRNA levels were unresponsive to increased testosterone concentration, but, unlike SGP-2 mRNA, transferrin mRNA increased as germ cells were restored after testosterone administration. These results suggest that SGP-2 mRNA is not regulated by testosterone or germ cells, but that the level of transferrin mRNA is influenced by the interaction of Sertoli and germ cells in the adult rat testis.

Maintenance of advanced spermatogenic cells in the adult rat testis: quantitative relationship to testosterone concentration within the testis.

The studies described herein were designed to examine whether there is a threshold concentration of testosterone (T) within the seminiferous tubules that is required to maintain spermatogenesis in the rat, or alternatively, whether there is a dose-response relationship between the intratesticular T concentration and the maintenance of spermatogenesis. T was administered to intact adult male rats via sustained release polydimethylsiloxane capsules in order to experimentally clamp T at well defined concentrations within the seminiferous tubules. Implantation of T-filled capsules of increasing sizes resulted in linear increases in T concentrations in serum, interstitial fluid, and seminiferous tubule fluid (STF). We examined the effect of step decreases in intratesticular T concentration on the numbers of advanced spermatogenic cells maintained by the testis over a 2-month period. Quantitatively complete spermatogenesis was maintained despite an 80% reduction in the STF T concentration (to approximately 13 ng/ml) from control values. The ability of the testis to maintain complete spermatogenesis was extremely sensitive to further decreases in STF T concentration. Thus, reduction of the STF T concentration from approximately 13 to 9 ng/ml resulted in a reduction in the number of advanced spermatids that were maintained in the testis by approximately 100 x 10(6). Reduction of the STF T concentration to approximately 4 ng/ml resulted in a further reduction in the number of advanced spermatids per testis by 100 x 10(6). Taken together, these data support the contention that there is far more T present within the seminiferous tubules of intact rat testes than is required to maintain quantitatively normal spermatogenesis and reveal for the first time that there is a dose-response relationship between the STF T concentration and the quantitative maintenance of advanced spermatogenic cells in the rat testis.

Exogenously administered testosterone maintains spermatogenesis quantitatively in adult rats actively immunized against gonadotropin-releasing hormone.

The administration of testosterone via Silastic capsules has been shown previously to maintain advanced spermatid number quantitatively in intact rats in which LH but not FSH was suppressed, but not in hypophysectomized rats, indicating that pituitary factors in addition to LH are required for the quantitative maintenance of spermatogenesis in the rat. The objective of the present study was to examine whether testosterone is capable of maintaining quantitatively normal spermatogenesis in rats in which both LH and FSH are suppressed. Intact adult male rats were actively immunized against GnRH by intradermal injection of GnRH conjugated to human serum globulin; control rats received intradermal injections of saline and adjuvant. Four weeks after the primary immunization, GnRH-immunized rats received the first booster injection and, at the same time, received testosterone-filled polydimethylsiloxane (PDS) implants of 4, 8, 12, or 24 cm or empty implants. Booster injections were repeated every 2 weeks for 8 weeks. At that time, rats were killed, and serum levels of LH, FSH, and testosterone, testicular advanced spermatid number, and seminiferous tubule fluid testosterone concentrations were determined. Four weeks after the initial administration of GnRH immunogen, i.e. before the first booster injection, serum levels of testosterone, LH, and FSH and the number of advanced spermatids per testis were not different from those in controls. Eight weeks after the first booster injection, serum LH and FSH and advanced spermatids were undetectable in all GnRH-immunized rats. The administration of testosterone-filled PDS implants of 4 and 8 cm to GnRH-immunized rats for 8 weeks resulted in the maintenance of 105 +/- 6 and 161 +/- 5 x 10(6) advanced spermatid/testis, respectively, significantly less than the control value (237 +/- 19 x 10(6)). In GnRH-immunized rats that received testosterone-filled PDS implants of 12 or 24 cm, the advanced spermatid numbers per testis (228 +/- 4 and 229 +/- 8 x 10(6), respectively) were not significantly different from those in controls. These results indicate that testosterone is capable of maintaining spermatogenesis quantitatively in the adult rats testis, in the absence of both radioimmunoassayable LH and FSH.

Restoration of advanced spermatogenic cells in the experimentally regressed rat testis: quantitative relationship to testosterone concentration within the testis.

We examined the effect of exogenously administered testosterone (T) on the quantitative restoration of advanced spermatogenic cells in adult rat testes rendered azoospermic by treating rats with polydimethylsiloxane (PDS) implants of T and estradiol (E). Experimental rats received PDS-TE implants for an initial 8-week period; control rats received empty implants. By 8 weeks of PDS-TE treatment, rats became severely oligospermic, and the T concentration within the seminiferous tubule fluid (STF) was reduced approximately 80% (from 57.8 ng/ml in controls to 9.6 ng/ml). After the initial 8-week PDS-TE treatment, PDS-TE implants were removed from one group of rats; a second group of PDS-TE-implanted rats received an additional PDS-T implant of 24 cm. Eight weeks after the removal of PDS-TE implants or the implantation of additional T, testis weight and numbers of advanced spermatogenic cells were restored to those of control rats. The STF T concentration 8 weeks after the removal of PDS-TE implants also was restored to that in control rats. In contrast, the STF T concentration increased to only 40% of control values in the rats that received an additional T implant. Despite this 60% reduction in T concentration compared to the control value, advanced spermatogenic cell number was restored to a value indistinguishable from that of intact controls. These observations indicate that spermatogenesis can be quantitatively restored in PDS-TE-implanted rats with exogenously administered T, and moreover, that this restoration does not require the high T concentration found in the STF of intact control rats.

Quantitative restoration of advanced spermatogenic cells in adult male rats made azoospermic by active immunization against luteinizing hormone or gonadotropin-releasing hormone.

The ability of testosterone to quantitatively restore spermatogenesis in rats made azoospermic by active immunization against LH or GnRH was examined. Sexually mature adult male rats (n = 15/group) were actively immunized against ovine LH or GnRH-human serum globulin conjugate, while control rats (n = 10) were injected with saline. After 10 weeks of immunization, five rats per group were euthanized. For each rat, trunk blood was collected for determination of LH, FSH, and testosterone by RIA; seminiferous tubule fluid (STF) was collected from one testis per rat, and testosterone concentration was measured by RIA; the number of advanced spermatids per testis was determined from the contralateral testis. The results obtained after 10 weeks of treatment were as follows. 1) Serum LH and FSH were undetectable by RIA in GnRH-immunized rats. 2) Serum testosterone was undetectable in both the LH- and GnRH-immunized groups. 3) The testosterone concentration in STF (STF-T) was reduced from the control value of about 64 ng/ml to about 2 ng/ml in the LH- and GnRH-immunized rats. 4) LH- and GnRH-immunized rats were azoospermic. After the initial 10-week treatment period, five rats in each of the LH- and GnRH-immunized groups received 24-cm testosterone-filled polydimethylsiloxane (PDS-T) capsules (3 x 8 cm long) sc. The remaining immunized rats (n = 5/group) received empty capsules. Two months later, all rats were euthanized. Testis weights, serum testosterone, and STF-T concentrations remained significantly reduced in LH- and GnRH-immunized rats that did not receive testosterone supplementation, and the rats remained azoospermic. STF-T concentrations rose significantly (P less than 0.05) in the LH- and GnRH-immunized rats that received PDS-T, but were still significantly less (by approximately 80%) than the concentration in intact controls. Nonetheless, implantation of PDS-T caused restoration of advanced spermatogenic cells in the testes of both LH- and GnRH-immunized rats to numbers that were not significantly different from the number in controls. These data indicate that 1) testosterone is capable of quantitatively restoring spermatogenesis in rats actively immunized against LH or GnRH, suggesting that FSH may not be required for the restoration of spermatogenesis in adult rats; and 2) quantitatively complete restoration of spermatogenesis can occur at STF-T concentrations that are significantly reduced compared to those in intact controls.

To what extent can spermatogenesis be maintained in the hypophysectomized adult rat testis with exogenously administered testosterone?

In a previous study it was demonstrated that spermatogenesis can be maintained quantitatively with exogenously administered testosterone in adult intact rats that lack LH. The studies described herein were designed to examine the extent to which spermatogenesis can be maintained quantitatively with exogenously administered testosterone in adult rats that lack all pituitary hormones. Adult male rats were hypophysectomized and testosterone was administered at the time of hypophysectomy via sustained release polydimethylsiloxane (PDS) capsules of increasing lengths. We used the PDS capsules to clamp testosterone at defined concentrations within the seminiferous tubule fluid over a 2- to 3-month treatment period. Mean testis weights and advanced spermatid numbers per testis stabilized by 8 weeks of testosterone treatment regardless of testosterone dose. Both testis weight and advanced spermatid number responded to testosterone dose, reaching plateaus of 1.2 g and 170 x 10(6) per testis, respectively. These values were 60% of, and significantly less than, the respective control values. This result was in striking contrast to the results of our previous study of LH-suppressed intact rats, in which exogenously administered testosterone resulted in testis weights and advanced spermatid numbers that plateaued at values not significantly different from those in controls. These different effects of testosterone in intact and hypophysectomized rats occurred despite the fact that the seminiferous tubule fluid testosterone concentrations achieved in the hypophysectomized rats (up to 25 ng/ml) were greater than the minimal testosterone concentration found previously to be required to maintain spermatogenesis quantitatively in LH-suppressed intact rats (13 ng/ml). Taken together, these results demonstrate clearly that intratesticular testosterone doses that are as high as or higher than those that maintain spermatogenesis quantitatively in intact rats lacking LH fail to maintain spermatogenesis quantitatively in rats lacking all pituitary hormones.

Restoration of spermatogenesis by exogenously administered testosterone in rats made azoospermic by hypophysectomy or withdrawal of luteinizing hormone alone.

The major objective of the studies presented herein was to compare the extent to which exogenously administered testosterone is able to restore spermatogenesis in adult rats made azoospermic by withdrawal of all pituitary hormones (hypophysectomy for 4 weeks) vs. withdrawal of LH alone [testosterone- and estradiol-filled (TE) polydimethylsiloxane implants of 2.5 and 0.1 cm, respectively, for 8 weeks]. In hypophysectornized (Hypox) rats, serum LH and FSH were both undetectable; in the rats that received TE implants, serum LH was undetectable, but FSH was unaffected compared to control values. Seminiferous tubule fluid testosterone concentrations were reduced significantly from their control values of 60-65 to 1.4-1.7 ng/ml in the azoospermic Hypox and TE rats. These rats then received testosterone-filled implants of 4, 12, 18, or 24 cm and were killed 2 months later. In both the Hypox and TE rats, seminiferous tubule fluid testosterone concentrations rose linearly with increasing capsule sizes, and with each of the implant sizes, these concentrations did not differ significantly between the Hypox and TE rats. This made it possible for the first time to examine the effects of comparable intratesticular testosterone concentrations on the numbers of advanced spermatids per testis that could be restored in the azoospermic testes of rats lacking all pituitary factors vs. those lacking only LH. The results that we present demonstrate that the numbers of restored advanced spermatids were consistently and significantly lower in Hypox than in TE rats despite equivalent seminiferous tubule fluid testosterone concentrations. These results provide quantitative conclusive evidence to support the contention that pituitary factors in addition to LH are required for the quantitative restoration of spermatogenesis in the adult rat testis.

Temporal gene expression is restored concomitantly with germ cells in the experimentally regressed rat testis.

The present study was designed to examine the effect of hypophysectomy and subsequent testosterone administration on germ cell numbers and germ cell- and Sertoli cell-specific mRNA levels in adult rats. Rats were hypophysectomized and 4 weeks later received 24-cm testosterone-containing polydimethylsiloxane (PDS) implants. Sham-hypophysectomized rats received an empty PDS implant. At 0 and 3 days, and at 1, 2, 4, and 8 weeks, rats were killed. One testis from each rat (n = 4/group) was used to prepare total RNA; the other testis was used to enumerate stage VII-VIII germ cells. cDNA probes for germ cell and Sertoli cell products were used to monitor germ cell- and Sertoli cell-specific mRNAs on Northern blots. Four weeks after hypophysectomy (0 days), preleptotene and pachytene spermatocytes and round and elongating spermatids were reduced in number to 54%, 12%, 1%, and 0%, respectively, of the control values. Testosterone administration caused a time-dependent increase in germ cell numbers; after 8 weeks of testosterone treatment, preleptotene and pachytene spermatocytes and round and elongating spermatids were 75%, 79%, 74%, and 22%, respectively, of control values. Lactate dehydrogenase-C, phosphoglycerate kinase-2, protamine-1, and sulfated glycoprotein-2 mRNA levels (on a per micrograms RNA basis) were 34%, 34%, less than 1%, and 580% of control values, respectively, 4 weeks after hypophysectomy and 79%, 87%, 61%, and 192% of control values, respectively, after 8 weeks of testosterone treatment. Pachytene spermatocyte and round spermatid numbers increased, while Sertoli cell sulfated glycoprotein-2 mRNA levels decreased, with respect to 4 week hypophysectomy values, as early as 3 days after implantation of testosterone capsules. In contrast, germ cell (lactate dehydrogenase-C, phosphoglycerate kinase-2, and protamine-1) mRNA levels increased to the greatest extent between 1-4 weeks after the start of testosterone treatment and, after a short lag period, reflected increases in germ cell type and number. The results indicate that cell-specific mRNAs appear concomitantly with germ cell reappearance in a time-dependent manner in the testes of testosterone-treated hypophysectomized adult rats.

Testicular endocrine function in GH receptor gene disrupted mice.

The consequences of disruption of GH receptor gene in GH receptor knockout mice on testicular function were evaluated. Adult male GH receptor knockout mice and their normal siblings were divided in to two subgroups and treated with either saline or ovine LH (0.3 microg/g BW) in saline. One hour after saline or LH administration, blood was obtained via heart puncture. Plasma IGF-I, LH, FSH, PRL, androstenedione, and testosterone levels were measured by RIAs. Testicular LH and PRL receptor numbers as well as pituitary LHbeta-subunit and testicular sulfated glycoprotein-2 mRNA levels were measured. Also, testicular morphometric analysis was performed. Unlike in normal, wild-type mice, the circulating IGF-I was undetectable in GH receptor knockout mice. The plasma PRL levels were (P<0.01) higher in GH receptor knockout mice than in their normal siblings. The basal LH secretion was similar in normal and GH receptor knockout mice. However, the circulating FSH levels were lower (P<0.001) in GH receptor gene disrupted mice. Administration of LH resulted in a significant (P<0.001) increase in plasma testosterone levels in both GH receptor knockout and normal mice. However, this testosterone response was attenuated (P < 0.01) in GH receptor knockout mice. Plasma androstenedione responses were similar in both GH receptor knockout and normal mice. Testicular LH and PRL receptor numbers were significantly decreased in GH receptor knockout mice. The results of the morphometric analysis of the testis revealed that the Leydig cell volume per testis was reduced in mice with GH receptor gene disruption. The steady-state of LHbeta-subunit and testicular sulfated glycoprotein-2 mRNA levels were not different in GH receptor knockout mice relative to their normal siblings. The present in vivo study demonstrates that in GH receptor knockout mice, LH action on the testis in terms of testosterone secretion is significantly attenuated and suggests that this is due to a decrease in the number of testicular LH receptors. The reduced number of PRL receptors may contribute to the diminished responsiveness of testicular steroidogenesis to LH by decreased ability to convert androstenedione to testosterone. These changes are most likely due to the absence of circulating IGF-I. These findings provide evidence that systemic IGF-I plays a major modulatory role in testicular endocrine function.

Ultrasound-guided transcervical tubal catheterization for assisted reproduction: a learning program using laparoscopy for confirmation.

The result of this pilot study confirmed that the US-directed transcervical tubal catheterization procedure for assisted reproduction can be learned over a short period of time and may well produce comparable PRs as seen with laparoscopic transfer. However, practice in the technique with confirmation of placement by laparoscopy is advised before incorporating this procedure into a program of assisted reproduction.

Immunoneutralization of gonadotropin-releasing hormone and subsequent treatment with testosterone Silastic implants in rats: an approach toward developing a male contraceptive.

STUDY OBJECTIVE: To determine the extent to which increasing doses of exogenous testosterone (T) administered via Silastic implants can restore spermatogenesis and fertility to rats made azoospermic by active immunization against gonadotropin-releasing hormone (GnRH). DESIGN: Male rats were made azoospermic by active immunization against GnRH. Increasing doses of exogenously administered T (via Silastic implants) were administered for 8 weeks, and testicular sperm concentration and ability to impregnate female rats were evaluated. SETTING: Reproductive Endocrinology Laboratory, Department of Obstetrics and Gynecology, University of Colorado Health Sciences Center, Denver, Colorado. ANIMALS: Sexually mature male Sprague Dawley rats (SASCO, Omaha, NE). RESULTS: Suppression of gonadotropins and azoospermia was achieved by actively immunizing rats against GnRH. Testosterone was capable of restoring quantitatively complete spermatogenesis and fertility in GnRH-immunized azoospermic rats. This relationship was dose-dependent, as evidenced by the partial restoration of spermatogenesis and fertility observed in animals replaced with smaller T Silastic implants. CONCLUSION: Gonadotropin-releasing hormone immunization and T-filled Silastic implants may provide a model to study isolated gonadotropin deficiency and for the development of a reversible male contraceptive.

Preprogrammed, unmonitored ovarian stimulation reduces expense without compromising the outcome of assisted reproduction.

OBJECTIVE: To determine if a novel, preprogrammed, unmonitored stimulation protocol could reduce the cost of assisted reproductive technology (ART) without compromising outcome or safety. DESIGN: Prospective, nonrandomized study of unmonitored ART versus traditional monitoring. SETTING: University ART program. PATIENT(S): Infertile women aged < 39 years, with a basal FSH level < 15 mIU/mL (conversion factor to SI unit, 1.00) and regular menstrual cycles, undergoing ART. INTERVENTION(S): Oocyte retrieval was performed at a predetermined time in 72 unmonitored cycles based on age and basal FSH level. No monitoring of any type was performed before retrieval. There were 86 monitored control cycles. MAIN OUTCOME MEASURE(S): The number of oocytes, and embryos; complications including ovarian hyperstimulation. RESULT(S): The total cost for unmonitored ART was significantly less than for monitored cycles. There was no difference between groups for patient age, number of oocytes obtained, or number of metaphase II oocytes. For non-male-factor patients, the number of oocytes fertilized, number of embryos transferred, and the clinical pregnancy rates were comparable. There was one case of severe hyperstimulation requiring hospitalization in the unmonitored group. CONCLUSION(S): This novel, unmonitored ovarian stimulation protocol provides ART at a significantly lower cost than is incurred with traditional monitoring, with no apparent compromise in outcome.

Hydrosalpinx treated with extended doxycycline does not compromise the success of in vitro fertilization.

OBJECTIVE: To determine if extended treatment with doxycycline before and after an in vitro fertilization (IVF) procedure can minimize the detrimental effect of a hydrosalpinx. DESIGN: Retrospective analysis. SETTING: University IVF program. PATIENT(S): Patients undergoing IVF, including 17 with a hydrosalpinx, 25 with adhesions or proximal tubal occlusion, and 22 with endometriosis or unexplained infertility. INTERVENTION(S): Women with a documented hydrosalpinx were prescribed doxycycline 100 mg twice daily starting 1 week before expected retrieval and continued until 6 days after retrieval. No antibiotics were prescribed in the other groups. MAIN OUTCOME MEASURE(S): Implantation rates and IVF outcomes. RESULT(S): Implantation rates were 30% for the doxycycline-treated group of patients with a hydrosalpinx, 27% for the group with tubal occlusion/adhesion, and 24% for the group with endometriosis or unexplained infertility. Eight (47%) of 17 patients with a hydrosalpinx had a live birth, compared with 11 (44%) of 25 for the group with tubal occlusion/adhesion and 12 (55%) of 22 for the group with endometriosis/unexplained infertility. There were no differences between the groups in patient age, number of oocytes retrieved, fertilization rate, or number of blastomeres of the transferred embryos. CONCLUSION(S): No detrimental effect of a hydrosalpinx was evident for patients treated with extended doxycycline. Tremendous cost savings can be realized if treatment with 2 weeks of an inexpensive antibiotic provides outcomes comparable to surgical correction of a hydrosalpinx before IVF.

Application of the cavitron ultrasonic surgical aspirator (CUSA) for gynecological laparoscopic surgery using the rabbit as an animal model.

OBJECTIVE: To study the potential application of the cavitron ultrasonic surgical aspirator (CUSA) in gynecological laparoscopic surgery using a rabbit animal model. DESIGN: Twenty-six rabbits were prospectively randomized into two groups. Laparoscopically directed standard injuries were made on the randomly assigned horn and sidewall in all animals with the CUSA. Contralateral injuries were made with a contact neodymium-yttrium aluminum garnet (Nd:YAG) laser in group 1 and with bipolar cautery in group 2. Adhesion and inflammation scores were assessed for two animals in each group at 24, 48, and 72 hours, and seven animals in each group at 14 days. SETTING: University animal research facility. MAIN OUTCOME MEASURES: Adhesion and inflammation scores were compared between animals in the CUSA versus Nd:YAG study and the CUSA versus bipolar cautery at 14 days. RESULTS: No significant difference in uterine or sidewall adhesion scores was noted between the CUSA versus Nd:YAG or the CUSA versus bipolar cautery. Bipolar cautery produced significantly less inflammation on the uterine horn compared with the CUSA (3.0 +/- 0.2 versus 5.3 +/- 0.7, P = 0.0001), but no difference in sidewall inflammation was noted between the CUSA compared with bipolar cautery. No difference in inflammation was observed between the CUSA and the Nd:YAG laser. CONCLUSIONS: The bipolar cautery appears to be preferable to the CUSA for coagulation of uterine lesions, although dissection of the uterus is not possible with bipolar cautery. The CUSA and the Nd:YAG appear to be comparable for uterine horn dissection. Because the CUSA causes similar adhesion formation and tissue inflammation at the sidewall when compared with the Nd:YAG laser and bipolar cautery and may be less likely to damage blood vessels, ureters, or other collagen-rich tissues, the CUSA may represent a promising new surgical tool for laparoscopically directed peritoneal dissection.

Reproductive sequelae in female rats after in utero and neonatal exposure to the phytoestrogen genistein.

OBJECTIVE: To determine reproductive sequelae in female rats after in utero and lactational dietary exposure to genistein. DESIGN: Experimental animal study. SETTING: University laboratory. ANIMAL(S): Sprague Dawley rats. INTERVENTION(S): Pregnant rats were fed control rat chow or rat chow incorporated with genistein (approximately 50 microg/d) beginning on day 17 of gestation and continuing until the end of lactation (postpartum day 21). Genistein-exposed female pups were divided into two groups on day 21. One group continued to receive a genistein-added diet (G70); the other group was changed to a control diet (Ex-G). At necropsy (days 21 and 70), blood and reproductive tissues were collected. MAIN OUTCOME MEASURE(S): Serum levels of gonadotropins and gonadal steroids and histopathologic examination of the ovaries. RESULT(S): The weight of the ovaries and uterus and serum levels of E2 and progesterone in genistein-exposed rats on day 21 (G21) were significantly reduced compared with control rats. On day 70, serum levels of E2, progesterone, LH, and FSH were similar in all groups. Atretic follicles and secondary interstitial glands were more common in G70 and Ex-G rats compared with control rats. Cystic rete ovarii was observed in some G70 and Ex-G rats. CONCLUSION(S): Our data indicate that in utero and lactational exposure to dietary genistein adversely affects reproductive processes in the adult female rat.

The effects of chronic administration of pyrimethamine on spermatogenesis and fertility in male rats.

The present study examines whether the antifertility effects of pyrimethamine (PYR), an inhibitor of dihydrofolate reductase, are mediated by a reduction in intratesticular testosterone (T) concentrations or whether PYR exerts its effect by a cytotoxic insult to spermatogenic cells that is independent of intratesticular testosterone. Adult male rats were treated daily with 100 mg/kg (n = 16) or 400 mg/kg (n = 16) of PYR in honey for 8 weeks. Control rats (n = 16) received honey without PYR. Eight weeks after treatment, five rats from each PYR-treated group and five control rats were mated with normal cycling female rats, and fertility was assessed. These rats were euthanized after the fertility trial; testis weight, testicular sperm, and epididymal sperm counts were determined, and serum levels of T, LH, FSH, and seminiferous tubule fluid T (STF-T) concentrations were measured by RIA. Testes from three rats per group were perfusion-fixed for histological evaluation. PYR was discontinued in the remaining rats for 8 weeks and similar parameters were evaluated after 8 weeks of recovery. PYR (100 mg/kg/day) treatment for 8 weeks did not have any effects on organ weights, testicular and epididymal sperm counts, and hormone levels when compared to controls. In contrast, PYR (400 mg/kg/day) treatment significantly reduced testis and epididymis weights, testicular and epididymal sperm counts, and fertility. Despite these effects, serum T, LH, FSH, and STF-T concentrations were not altered.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in testicular morphology in boars actively immunized against gonadotropin hormone-releasing hormone.

Alterations in testicular morphology were studied in boars actively immunized against gonadotropin hormone releasing hormone (GnRH). Ten boars were divided equally into two experimental groups (five GnRH-immunized, and five controls). Antibody production was achieved by conjugating GnRH to human serum globulin (hSG). The GnRH-hSG conjugate was emulsified in complete Freund's adjuvant, and administered to boars at 12 weeks of age. Boars were given a booster in incomplete Freund's adjuvant on week 18 and 20. The presence of high antibody titers to GnRH caused luteinizing hormone and testosterone to decline to nondetectable levels. Morphometric examination showed a reduction in percentage volume in Leydig cells/unit testis, seminiferous tubule diameter and seminiferous epithelial height, and an increase in non-Leydig cell interstitial tissue in GnRH-immunized boars compared with controls. Histologic evaluation displayed severe damage of the seminiferous epithelium, absence of spermatids, incomplete cell associations, disruption of Sertoli cells, formation of multinucleated giant cells, and a striking reduction in size and cytoplasmic structures of Leydig cells in GnRH-immunized animals. These results demonstrate the potent inhibitory effects of GnRH immunoneutralization on the boar reproductive system.

Suppression of growth hormone does not affect ongoing spermatogenesis in rats.

Recent evidence suggests that growth hormone (GH) may enhance physiologic processes, such as spermatogenesis, in addition to causing classical anabolic effects. We have previously shown that testosterone restores spermatogenesis in rats that were made azoospermic by immunization against gonadotropin-releasing hormone (GnRH). In this study, we investigated whether suppression of GH affects spermatogenesis and the ability of testosterone to restore spermatogenesis following immunization against GnRH and/or growth hormone-releasing hormone (GHRH). Twelve rats were actively immunized against GnRH (anti-GnRH), twelve rats were actively immunized against GHRH (anti-GHRH), six rats were immunized against both GnRH and GHRH (anti-GnRH/GHRH), and six rats served as controls. Two weeks after the second booster, six rats each from the anti-GnRH and anti-GHRH groups as well as the six anti-GnRH/GHRH rats received 24-cm testosterone-filled Silastic implants (T), and the remaining six rats from each of these groups received empty Silastic implants. All rats were euthanized 2 months later. Weights of testes and testicular sperm counts were determined. Serum testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), growth hormone (GH), and insulin-like growth factor-1 (IGF-1) concentrations were determined by radioimmunoassays. Serum GH and IGF-1 were suppressed in anti-GHRH rats. IGF-1 was partially restored by testosterone in anti-GHRH and in anti-GnRH/GHRH rats, but GH was restored to control value in anti-GnRH/GHRH rats. Serum LH and FSH were suppressed in anti-GnRH and anti-GnRH/GHRH rats, but only FSH was partially restored by testosterone. Suppression of GH did not affect maintenance of spermatogenesis. However, because T partially restored GH and IGF-1 levels in anti-GnRH/GHRH rats and because spermatogenesis was found to be restored in these rats, we conclude that GH does not play a role in the maintenance of spermatogenesis in adult rats, but it may be required for the replenishment of germ cells in experimentally induced regressed rat testes.

Does ethane 1,2-dimethanesulphonate (EDS) have a direct cytotoxic effect on the seminiferous epithelium of the rat testis?

The authors examined the possibility that ethane 1,2-dimethanesulphonate (EDS) has a cytotoxic effect on spermatogenesis that is not secondary to androgen withdrawal resulting from the well known cytotoxic effect of EDS on Leydig cells. Adult male rats were implanted with polydimethylsiloxane (PDS) capsules containing testosterone (T) and estradiol (E), and were simultaneously injected with EDS. The PDS-TE implants, by inhibiting luteinizing hormone (LH) production, prevented Leydig cells from repopulating the testis and clamped testosterone within the seminiferous tubules at increasing concentrations relative to implant size. In rats that received EDS alone, the number of advanced spermatids per testis was significantly reduced by 2 weeks, but within 8 weeks returned to the numbers maintained in vehicle-injected control rats or in vehicle-injected rats that received testosterone- and estradiol-filled capsules of 24 cm and 0.1 cm, respectively (PDS-24TE). Surprisingly, in rats that received an EDS injection plus PDS-24TE implants, the number of advanced spermatids per testis was significantly reduced at 8 weeks and severe seminiferous tubule atrophy occurred despite the fact that the testosterone concentration was sufficient to quantitatively maintain spermatogenesis in vehicle-injected rats. In rats injected with EDS and implanted with 24 cm testosterone but not estradiol-filled capsules (PDS-24T), the advanced spermatid number per testis was significantly higher than that in the EDS plus PDS-24TE rats, but significantly lower than that in control rats. These results suggest that EDS may have a cytotoxic effect on the seminiferous epithelium that is independent of the elimination of Leydig cells, and the EDS and estradiol act synergistically to exert a profound toxic effect on spermatogenesis.

Transcervical gamete and zygote intrafallopian transfer. Does it enhance pregnancy rates in an assisted reproduction program?

OBJECTIVE: To evaluate the role of early tubal transfer procedures, we compared outcomes of transcervical gamete intrafallopian transfer (TC-GIFT) and transcervical zygote intrafallopian transfer (TC-ZIFT) versus in vitro fertilization/embryo transfer during the first two years of our assisted reproduction (AR) program. STUDY DESIGN: Prospective, nonrandomized, concurrent, controlled comparison of TC-GIFT and TC-ZIFT pregnancy outcomes versus those after IVF-ET. All cycles for patients less than age 39 undergoing transfer of at least three viable oocytes, zygotes or embryos in the first two years of our program were included. Patients with normal fallopian tubes underwent TC-GIFT (n = 9) or TC-ZIFT (n = 12), whereas those with tubal compromise underwent IVF-ET (n = 28). RESULTS: Implantation rates were 4.2% for TC-ZIFT, 2.8% for TC-GIFT and 3.7% for combined TC procedures as compared to 7.4% for IVF-ET. Delivery rates were no different for the TC procedures than the IVF-ET procedures (14%). Patients ages, number of oocytes retrieved and number transferred were comparable between the TC and IVF-ET groups. CONCLUSION: TC-GIFT and TC-ZIFT did not enhance the pregnancy outcome as compared to IVF-ET in the first two years of our AR program. Ultrasound-directed tubal catheterization is harder to learn and more difficult and expensive to perform than simple uterine embryo transfer. Since we could not demonstrate an improved outcome for TC transfers even in a new AR program, IVF-ET and laparoscopic GIFT are now our procedures of choice.