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Prenatal androgenization induces a polycystic ovary syndrome-like phenotype in adult female offspring, which is associated with alterations that can be detected in the fetal ovary, suggesting gestational origins of this condition. We therefore investigated whether increased prenatal androgen exposure also altered testicular development using ovine animal models. Biweekly maternal testosterone propionate (TP; 100 mg) from day 62 to day 70/day 90 of gestation altered male developmental trajectory. In male fetuses serum LH was decreased (P < .01), and testicular STAR, CYP11, and CYP17 abundance were reduced. Coincident with this, basal testicular T synthesis was decreased in vitro (P < .001). Leydig cell distribution was severely perturbed in all testes prenatally exposed to TP (P < .001). To examine the contribution of estrogens, fetuses were injected with TP (20 mg), the potent estrogen agonist, diethylstilbestrol (DES; 20 mg), or vehicle control at day 62 and day 82 and assessed at day 90. The effects of fetal (direct) TP treatment, but not DES, paralleled maternal (indirect) TP exposure, supporting a direct androgen effect. Cessation of maternal androgenization at day 102 returned Leydig cell distribution to normal but increased basal T output, at day 112, demonstrating Leydig cell developmental plasticity. Earlier maternal androgen exposure from day 30 similarly influenced Leydig cell development at day 90 but additionally affected the expression of Sertoli and germ cell markers. We show in this study that increased prenatal androgen exposure alters development and function of Leydig cells at a time when androgen production is paramount for male development. This supports the concept that gestational antecedents associated with polycystic ovary syndrome may have effects on the male fetus.

Prolactin (PRL) is mainly expressed in the pituitary in rodents, whereas in humans, expression is observed in many extrapituitary sites, including lymphocytes. Due to the lack of adequate experimental models, the function of locally produced PRL in the immune system is largely unknown. Using transgenic rats that express luciferase under the control of extensive human PRL regulatory regions, we characterized immune cell responses to thioglycollate (TG)-induced peritonitis. Resident populations of myeloid cells in the peritoneal cavity of untreated rats expressed barely detectable levels of luciferase. In contrast, during TG-induced peritonitis, cell-specific expression in both neutrophils and monocytes/macrophages in peritoneal exudates increased dramatically. Elevated luciferase expression was also detectable in peripheral blood and bone marrow CD11b+ cells. Ex vivo stimulation of primary myeloid cells showed activation of the human extrapituitary promoter by TNF-α, lipopolysaccharide, or TG. These findings were confirmed in human peripheral blood monocytes, showing that the transgenic rat provided a faithful model for the human gene. Thus, the resolution of an inflammatory response is associated with dramatic activation of the PRL gene promoter in the myeloid lineage.

The seminal vesicles (SVs), like much of the male reproductive tract, depend on androgen-driven stromal-epithelial interactions for normal development, structure, and function. The primary function of the SVs is to synthesize proteins that contribute to the seminal plasma and this is androgen dependent. However, the cell-specific role for androgen action in adult SVs remains unclear. This study analyzed the SV in mice with targeted ablation of androgen receptors specifically in smooth muscle cells (PTM-ARKO) to determine in vivo whether it is androgen action in a subset of the SV stroma, the smooth muscle cells, that drives epithelial function and identity. These mice have significantly smaller SVs in adulthood with less smooth muscle and reduced epithelial cell height. Less epithelial cell proliferation was observed in adult PTM-ARKO SVs, compared with controls, and production of seminal proteins was reduced, indicating global impairment of epithelial cell function in PTM-ARKO SVs. None of these changes could be explained by altered serum testosterone or estradiol concentrations. We also demonstrate altered SV responsiveness to exogenous testosterone and estradiol in PTM-ARKO mice, indicating that smooth muscle androgen receptors may limit the SV epithelial proliferative response to exogenous estrogens. These results therefore demonstrate that the smooth muscle cells play a vital role in androgen-driven stromal-epithelial interactions in the SV, determining epithelial cell structure and function as well as limiting the SV epithelial proliferative response to exogenous estrogens., Androgen signaling via the smooth muscle cells is vital for normal seminal vesicle structure and function.

Inhibitor of differentiation (Id) proteins act during embryogenesis and development to repress gene transcription required for lineage commitment, while promoting cell growth. Growth factors belonging to the TGF beta superfamily of signaling molecules, notably the bone morphogenetic proteins (BMPs) and activin, can regulate Id expression in these tissues. Id expression and function in adult physiology is less well determined, and we hypothesized a role for Id proteins in the adult mammalian ovary. Immunohistochemistry for Id1, Id2, Id3, and Id4 in the sheep ovary revealed consistent expression in granulosa and thecal cells of ovarian follicles throughout development. In atretic follicles, Id proteins were selectively down-regulated in thecal cells (P < 0.0001). Additionally, Id1 was universally up-regulated in the cumulus cells adjacent to the oocyte. Immunohistochemistry for phospho (p)-smad 1/5/8 signaling components (stimulated by BMPs) showed a punctate pattern of expression whereas p-smad 2/3 (stimulated by activin) was ubiquitously expressed in follicles. Neither pathway, however, displayed differential staining in line with Id1 cumulus-specific expression, suggesting a more complex relationship between Id1 expression and TGF beta signaling in these cells. Nevertheless, in vitro, stimulation of ovine granulosa cells with BMP6 or activin A led to a respective increase and decrease in Id1 (P < 0.0001), Id2 (P < 0.0001), Id3 (P < 0.0001), and Id4 (P < 0.05) transcripts, and Id1 gene expression was further manipulated by the oocyte-secreted factors BMP15 and growth differentiation factor 9 (P < 0.001). These data confirm that TGF beta signaling can regulate Id gene expression in the sheep ovarian follicle and suggest a functional role for the Id family in the mammalian ovary. (Endocrinology 151: 1247-1256, 2010)

Within 2 days of birth, the mouse ovary is mainly composed of oocytes surrounded by a few pregranulosa cells forming primordial follicles that remain quiescent until they are recruited by intraovarian or other unknown factors to initiate growth of the oocyte and proliferation of the attendant granulosa cells. However, the role of the oocyte in this early development and organization of the follicle is poorly understood. The Dazl knockout (2/2) mouse in which there is total ablation of oocytes in fetal life has allowed us to address this issue. Ovaries from 2/2 females lack any follicular structure and have no cells positive for either Mullerian inhibiting factor or sulfated glycoprotein-1, indicating a lack of small follicles or corpora lutea. However, by immunocytochemistry, there are cells positive for 3bhydroxysteroid dehydrogenase, 17a-hydroxylase, and aromatase, indicating the presence of steroidogenically active cells capable of producing estrogen. This was confirmed by the presence of hypertrophied uterine endometrium expressing both estrogen receptor a (ERa) and ERb together with normal levels of plasma estradiol. In addition, these steroidogenically active cells contain ERb, inhibin a, and bBsubunits, and 2/2 mice have low measurable plasma inhibin A and B levels. The ovarian steroids and inhibins had no significant effect on either plasma or pituitary gonadotropin levels, with significantly (P , 0.01) lower LH and FSH in intact 1/1 and 1/2 females. However, significantly (P , 0.05) increased plasma inhibin B together with significantly (P , 0.05) lower FSH were observed in the 1/2 females. In conclusion, our data showed that despite oocyte loss in fetal life, the adult ovaries contained steroidogenically active cells capable of producing estradiol and inhibin. Furthermore, in the 1/2 mice, the enhanced plasma inhibin B implies a role for Dazl protein within the oocyte either from more small follicles or increased inhibin B production from each follicle. (Endocrinology 141: 4284 ‐ 4294, 2000)

The purpose of this study was to document the morphological changes in the seminiferous epithelium that underlie the compensatory testicular hypertrophy observed in response to unilateral orchidectomy (UO) in the adult rhesus monkey and to describe the concomitant response in the endocrine feedback loops controlling testicular function in this species. Adult male monkeys were implanted with indwelling venous catheters; seven animals were then subjected to UO (data are presented from six) and three to sham UO. Profiles of circulating concentrations of FSH, LH, testosterone (T), inhibin B, and pro-alpha-C were monitored in 12-h series of sequential blood samples collected before, on the day of UO (day 0), and on days 1, 2, 4, 8, 16, 32, and 42 or 43 after UO. In the UO monkeys, the remaining testis was taken on day 44. Sertoli and germ cells in the removed and remaining testes were counted and expressed either as number per testis or, in the case of the differentiated spermatogonia (B1, B2, B3, and B4), as number per cross-section of the seminiferous tubule. UO was associated with a marked increase in the number of all germ cells more mature than undifferentiated spermatogonia (Ap) in the remaining testis. Sertoli cell number, on the other hand, did not change, and it is therefore reasonable to propose that the primary locus of the spermatogenic compensation was the differentiated spermatogonia. The additional finding that the relationship between the number of Sertoli cells and total germ cells in the remaining testis became robust (r = 0.92; P 0.05 for the removed testis) indicated that in the monkey, spermatogenesis does not normally operate at its ceiling. The increased drive to the seminiferous tubule of the remaining testis is hypothesized to be mediated by the sustained increase in FSH secretion that was observed after UO, although a role for increased testicular T production cannot be excluded. The stimulus for increased FSH secretion was presumably provided by the abrupt, 50% decline in circulating inhibin B levels. Interestingly, inhibin B secretion by the remaining testis was not dramatically affected by UO, and therefore, the deficit in circulating levels of this hormone and thus the error signal to FSH secretion were maintained for the duration of the experiment. In contrast, the changes in circulating LH and T concentrations were only transient, and within 48 h of UO, these hormonal parameters had returned to control values. The mechanisms by which the remaining testis rapidly acquires the capacity to double T production in the face of an unchanging LH drive remains to be determined. The foregoing body of evidence suggests that sperm output by the monkey testis is regulated by the circulating concentration of FSH and that in physiological situations, FSH secretion is insufficient to stimulate spermatogenesis to its ceiling. The results also indicate that FSH secretion is controlled by a feedback system in which the feedforward arm (FSH-inhibin B) is less robust than the feedback loop (inhibin B-FSH). Thus, a decrease in the inhibin B feedback signal results in a sustained increase in FSH secretion that drives the testes toward their spermatogenic ceiling, which is presumably set by Sertoli cell number.

One of the hallmarks of polycystic ovary syndrome (PCOS) is increased ovarian androgen secretion that contributes to the ovarian, hormonal, and metabolic features of this condition. Thecal cells from women with PCOS have an enhanced capacity for androgen synthesis. To investigate whether this propensity is a potential cause, rather than a consequence, of PCOS, we used an ovine prenatal androgenization model of PCOS and assessed ewes at 11 months of age. Pregnant Scottish Greyface ewes were administered 100 mg testosterone propionate (TP) or vehicle control twice weekly from d 62 to 102 of gestation, and female offspring (TP = 9, control = 5) were studied. Prenatal TP exposure did not alter ovarian morphology or cyclicity, or plasma androgen, estrogen, and gonadotropin concentrations, at this stage. However, follicle function was reprogrammed in vivo with increased proportions of estrogenic follicles (P < 0.05) in the TP-exposed cohort. Furthermore, in vitro the thecal cells of follicles (>4 mm) secreted more LH-stimulated androstenedione after prenatal androgenization (P < 0.05), associated with increased basal expression of thecal StAR (P < 0.01), CYP11A (P < 0.05), HSD3B1 (P < 0.01), CYP17 (P < 0.05), and LHR (P < 0.05). This provides the first evidence of increased thecal androgenic capacity in the absence of a PCOS phenotype, suggesting a thecal defect induced during fetal life.

Androgen-driven stromal-epithelial interactions play a key role in normal prostate development and function as well as in the progression of common prostatic diseases such as benign prostatic hyperplasia and prostate cancer. However, exactly how, and via which cell type, androgens mediate their effects in the adult prostate remains unclear. This study investigated the role for smooth muscle (SM) androgen signaling in normal adult prostate homeostasis and function using mice in which androgen receptor was selectively ablated from prostatic SM cells. In adulthood the knockout (KO) mice displayed a 44% reduction in prostate weight and exhibited histological abnormalities such as hyperplasia, inflammation, fibrosis, and reduced expression of epithelial, SM, and stem cell identify markers (e.g. p63 reduced by 27% and Pten by 31%). These changes emerged beyond puberty and were not explained by changes in serum hormones. Furthermore, in response to exogenous estradiol, adult KO mice displayed an 8.5-fold greater increase in prostate weight than controls and developed urinary retention. KO mice also demonstrated a reduced response to castration compared with controls. Together these results demonstrate that prostate SM cells are vital in mediating androgen-driven stromal-epithelial interactions in adult mouse prostates, determining cell identity and function and limiting hormone-dependent epithelial cell proliferation. This novel mouse model provides new insight into the possible role for SM androgen action in prostate disease.

Exposure of a female fetus to increased androgens in utero results in an adult phenotype reminiscent of polycystic ovary syndrome. We investigated whether prenatal androgens could directly alter the structure and function of the fetal ovary. We examined fetal ovarian cell proliferation, germ cell volume, and the expression of steroid receptors and steroidogenic enzymes. In addition, we studied the inhibitors of differentiation (Ids) and the SLIT/Roundabout developmental pathways. Female fetuses were collected from ewes treated with 100 mg testosterone propionate (TP) or vehicle control (C), twice weekly from d 60 to 70 (C = 3, TP = 6) or d 90 (C = 6, TP = 8). Female fetuses were also collected at d 70 after a single injection of TP (20 mg) or vehicle C into the fetal flank at d 60 (C = 4, TP = 8). Prenatal androgenization had no effect on fetal ovarian morphology, cell proliferation, or germ cell volume. However, there was a reduction in the expression of StAR, CYP11A, CYP17, and LHR at d 90 of gestation. There was also an increase in Id1 immunostaining at d 90 and an increase in Id3 immunostaining at d 70. Direct injection of TP into the fetus down-regulated ovarian CYP11A, estrogen receptor α and β mRNA, and ROBO1 and up-regulated CYP19, androgen receptor immunostaining, and Id3 mRNA and protein. Although at d 90 prenatal androgenization does not result in structural changes of the fetal ovary, there are functional changes that may impact on ovarian development. TP has direct actions on the fetal ovary, and these may contribute to the adult ovarian phenotype in the ovine model of polycystic ovary syndrome.

Luteolysis in women is associated with an up-regulation of the expression and activity of matrix metalloproteinase-2 (MMP-2), which is inhibited by human chorionic gonadotropin (hCG) during maternal recognition of pregnancy. Because the primary source of MMP-2 is fibroblasts that do not express LH/hCG receptors, we aimed to investigate the regulation of MMP-2. Women with regular cycles having hysterectomy for nonmalignant conditions and women undergoing oocyte retrieval for assisted conception were used in this current study. Novel primary cultures and cocultures of luteinized granulosa cells and fibroblast-like cells in conjunction with human corpora lutea from different stages of the luteal phase were used to investigate the role of activin-A in the corpus luteum. The effect of hCG, activin-A, and follistatin on MMP-2 activity and expression was assessed by gelatin zymography and quantitative RT-PCR in primary cell cultures. Confirmation of signaling pathways involved in the activation of MMP-2 was assessed by immunofluorescence, RT-PCR, and quantitative RT-PCR. In primary cell culture, steroidogenic cells secrete activin-A and its inhibitors, inhibin-A and follistatin. Follistatin expression is up-regulated by hCG (P < 0.05). The fibroblast-like cells producing MMP-2 have the machinery for activin reception, expressing both type I and type II activin receptors and Smad proteins. Activin-A up-regulated both activity and expression of MMP-2 in fibroblast-like cells (P < 0.05). This activity was inhibited in cocultures of luteinized granulosa cells and fibroblast-like cells in the presence of hCG (P < 0.05) or follistatin (P < 0.01). Activin-A is an excellent candidate for an effector molecule in human luteolysis whose paracrine action is inhibited during maternal recognition of pregnancy.

FSH is secreted as a mix of isoforms with varying biologic attributes. To determine the functional significance of FSH heterogeneity, an acidic (ovine pituitary FSH; C-FSH) and less acidic mix (C-FSH exposed to neuraminidase; N-FSH) were administered to prepubertal lambs. Production of GnRH- induced less acidic FSH was blocked with a competitive GnRH receptor antagonist, Nal-Glu. Beginning 24 h after Nal-Glu, lambs were injected with C-FSH or N-FSH and LH. Controls included untreated, GnRH-treated, and Nal-Glu-treated groups. Blood samples were obtained at 2-h intervals. Plasma FSH levels were similar before treatment and increased over time in the C-FSH but not the N-FSH group (P0.001). Three of the six GnRH-treated ewes exhibited an LH surge. Peak E2 concentrations in the GnRH-treated animals were achieved 30-36 h after initiation of treatment. Peak circulating E2 levels tended to be higher in the C-FSH than in the GnRH-treated group. Only two of six N-FSH-treated ewes had a serum E2 rise. The C-FSH ewes had more estrogenic follicles than the GnRH and N-FSH groups (P0.05). Our findings show that C-FSH clears more slowly than N-FSH, and C-FSH is a better facilitator of follicular development and maturation than N-FSH.

In sheep, as in other mammalian species, the pronounced reduction in GnRH and gonadotropin secretion that characterizes stages of infertility is normally associated with a conspicuous increase in the secretion of PRL. A possible role of PRL in modulating gonadotropin release implies the presence and activation of specific receptors in target tissues (i.e. pituitary, hypothalamus). In this study, we investigated the expression of PRL receptor (PRL-R) messenger RNA (mRNA) in the sheep pituitary and the distribution of the translated product in specific pituitary cell types. Using primers designed to flank different regions of the extracellular and cytoplasmic domains of the PRL-R, two complementary DNA (cDNA) fragments, one of which was specific for the long-form PRL-R, were amplified by reverse transcriptase-PCR. Sequencing revealed more than 95% identity with nucleotides 267-1272 of the bovine PRL-R cDNA. When these cDNA fragments were used as probes for the detection of PRL-R mRNA expression by Northern analysis, three major transcripts of approximately 13, 10, and 3.5 kb were identified in the pituitary. Both probes detected identical transcripts, suggesting that primarily the long form of PRL-R is expressed in the sheep pituitary gland. No difference in the abundance of pituitary PRL-R mRNA transcripts was observed between anestrous and breeding season ewes (P > 0.05). Additional RT-PCR studies revealed the existence of a cDNA variant bearing a 39-bp insert with a premature stop codon. Translation of the PRL-R mRNA was confirmed by Western blot analysis. The identification of PRL-R in specific pituitary cell types was carried out by immunocytochemistry. Double immunofluorescent staining, using antibodies to the rat liver PRL-R and specific monoclonal antibodies to the LHbeta-subunit, FSHbeta-subunit, free alpha-subunit, PRL, or GH, revealed that in both the pars distalis and pars tuberalis, all pituitary cells expressing PRL-R immunoreactivity were positive for LHbeta, although only 53% of LHbeta-positive cells expressed PRL-R. A small proportion (2%) of gonadotrophs expressing PRL-R immunoreactivity were negative for FSHbeta, indicating the specific localization of PRL-R in LH (or LH/FSH) secreting cells. Further, a selective cytological association was detected in the pars distalis where LH gonadotrophs appeared surrounded by lactotrophs. In contrast to these observations, PRL-R immunoreactivity was completely absent in lactotrophs and in the vast majority (>98%) of somatotrophs. In conclusion, here we show the expression of PRL-R mRNA in the sheep pituitary and the specific translation of the signal in LH (or LH/FSH) gonadotrophs. These results support the hypothesis that PRL may be involved in the regulation of gonadotropin secretion through a paracrine mechanism within the pituitary gland and that this action does not seem to be mediated by changes in PRL-R mRNA expression.

An experiment was conducted in order to determine the pattern of, and the relationships between, the secretion of inhibin, estradiol, and androstenedione by the ovary and the concentration of LH, FSH, and PRL during the estrous cycle of sheep. The estrous cycles of 6 Finn-Merino ewes in which the left ovary had been autotransplanted to the neck were synchronized by two injections of cloprostenol (100 micrograms im) a potent analog of prostaglandin F2 alpha (PG) given 14 days apart. The ewes had ovarian and jugular venous blood samples taken at four hourly intervals from 42 h before the second PG injection until day 6 of the following cycle. All animals responded to PG with the preovulatory LH surge occurring within 58 +/- 2 h (mean +/- SEM). The concentration of FSH in jugular venous plasma fell (P less than 0.001) after the induction of luteolysis and then exhibited 3 peaks, the first coincident with the LH surge, the second on day 1, and the third on day 6. After injection of PG the secretion rates of inhibin, estradiol, and androstenedione increased (P less than 0.05) within 4-8 h. After this increase in the early follicular phase the secretion rate of estradiol continued to rise until the time of the LH surge (P less than 0.001). Although the secretion of androstenedione and inhibin increased in the 36 h before the LH surge the magnitude of this rise was less marked than for estradiol and was not statistically significant. Within 4-8 h of the start of the LH surge the secretion of estradiol and androstenedione declined rapidly reaching barely detectable levels within 16 h (P less than 0.001). In contrast the secretion of inhibin increased after the LH surge reaching a broad peak (P less than 0.05) of approximately 16-h duration, coincident with the second peak of FSH. From days 2-6 mean secretion of inhibin remained relatively stable at 2-6 ng/min although considerable variation was observed in individual profiles. The rate of estradiol secretion increased steadily from its nadir on day 1 to a broad peak centered around day 3 (3-6 ng/min, P less than 0.001) followed by a decline until by day 6 the estradiol secretion rate was less than 1 ng/min (P less than 0.01). The secretory profile for PRL showed a close relationship with estradiol secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Rabbit milk fat globule membranes have been shown to contain PRL receptors by a variety of criteria, including hormonal specificity and inhibition by specific anti-PRL receptor antisera. Collection of milk samples throughout the period of lactation facilitated a temporal study of the receptor content of the membrane fraction of these milk samples by Scatchard analysis after treatment with 5 M MgCl2 to dissociate endogenously bound PRL. Total receptor content was low after parturition (3.3 +/- 1.3 fmol/mg membrane protein) but increased subsequently, reaching maximal levels (43.7 +/- 3.4 fmol/mg) by day 21 of lactation. No significant difference in the Ka (4.9 X 10(9) +/- 0.35 M-1) of the milk receptor was detected over a 4-week suckling period. No apparent relation seemed to exist between rabbit serum PRL values measured by RIA in serum samples taken just before milking and milk PRL receptor content. Milk receptor content, however, was significantly (P < 0.02) correlated with the PRL receptor content of the gland when animals were sacrificed immediately after milking.

A highly specific heterologous double-antibody RIA has been developed to measure rabbit PRL by using guinea pig antiserum to human PRL and ovine [125 I]iodo-PRL. Rabbit pituitary PRL and serum give parallel dose-response curves in the assay and no cross-reaction (less than 0.1%) occurs with GH, placental lactogens, LH, FSH, or TSH from several different species. The assay is suitable for the measurement of human, ovine, bovine, caprine, and canine PRL in addition to rabbit PRL, but shows no cross-reaction with rat PRL. Reproducibility and precision of the assay are within acceptable limits. Gel filtration of rabbit pituitary PRL and rabbit serum on Sephadex G-100 revealed coincident peaks of activity measured by RIA and by PRL radioreceptor assay. The molecular weight of rabbit PRL appeared similar to that of ovine PRL. Serum PRL levels increased after the injection of both TRH and chlorpromazine and were reduced by CB154 (Bromocriptine). Venepuncture stress caused an increase in PRL in nonpregnant or postpartum non-suckled animals, but small or no increases were seen in lactating female rabbits.

To determine whether failure of the final stages of follicular maturation during anestrus in sheep was due to inadequate LH stimulation, two experiments were carried out in midanestrus, 1 yr apart, in six Finn-Merino ewes with an ovary or an ovary and uterus autotransplanted to the neck. In the first experiment, iv injections of ovine LH were given at a rate (one injection per 3 h for 24 h, one per 2 h for 24 h, and one per hour for 24 h) equivalent to the natural increase in frequency of LH pulses which occurs in the preovulatory period of the estrous cycle during the breeding season. In the second experiment, the frequency of LH pulses was kept constant at one per 3 h for 72 h. Each injection resulted in a pulse of LH which was qualitatively and quantitatively similar to an endogenous LH pulse. Jugular and ovarian venous samples were collected and assayed for LH, FSH, PRL, 17β-estradiol, and progesterone. An increase in the secretion of 17β-estradiol, a preovulatory LH peak, and normal corpus luteum fun...

By using a specific heterologous double-antibody RIA, changes in the blood levels of PRL during pregnancy, pseudopregnancy, and lactation have been investigated for the first time in the rabbit. Blood levels fluctuate during early and midpregnancy in a manner similar to that in pseudopregnancy. Levels decline in the third trimester of pregnancy and increase dramatically (3- to 25-fold) at or 1--2 days before delivery. Pituitary levels of PRL showed no significant alteration, and fetal serum and amniotic fluid levels of PRL remain low (less than 10 ng/ml) throughout pregnancy. No significant PRL-like, GH-like, or placental lactogen-like activity could be demonstrated either in serum or in extracts of placenta (n = 262) taken between days 10 and 31 of pregnancy. Postpartum blood levels of PRL were similar in lactating and postpartum nonlactating females. In lactating females, suckling evoked an immediate increase (15- to 25-fold) in circulating PRL levels. Handling the female or the iv injection of oxytocin during lactation did not cause PRL release. In contrast, manual test stimulation caused an immediate increase in blood levels of PRL and a response pattern very similar to that of natural suckling. These results suggest that PRL release during suckling occurs solely in response to the tactile stimulation of the teats.

High plasma levels of PRL induced by transplants of two donor pituitaries under the kidney capsule of adult male rats resulted in a prolonged suppression of plasma levels of LH and FSH although testosterone levels were maintained within normal limits. Castration of rats with pituitary transplants resulted in a normal though delayed rise in serum levels of both LH and FSH to levels equivalent to those in normal castrated controls. This increase in gonadotropin levels occurred in spite of maintenance of elevated PRL levels. Two experiments were carried out in which testosterone was restored after castration by Silastic testosterone-containing implants of various lengths (Exp 1:60, 30, and 10 mm; Exp 2: 30, 20, 10, 5, and 2 mm). In both experiments 60- and 30-mm testosterone implants prevented the postcastration rise in LH and FSH in both control and hyperprolactinemic rats. However, although the shorter testosterone implants delayed this rise in control rats, levels of LH and FSH increased by 4 days and were not significantly different from castrated rats without testosterone implants by 15 days after castration. In contrast, this rise in gonadotropins was abolished or considerably delayed by the shorter implants in hyperprolactinemic rats, demonstrating an increase in sensitivity of the hypothalamic pituitary axis to the negative feedback effects of testosterone in these animals. These results suggest that 1) to maintain suppression of gonadotropin secretion in hyperprolactinemia high levels of PRL alone are insufficient and gonadal steroids are required, and 2) high levels of PRL appear to sensitize the hypothalamic-pituitary axis to the negative feedback effects of gonadal steroids.