Pituitary and gonadal function in hypogonadotrophic hypogonadal (hpg) mice bearing hypothalamic implants

GnRH receptor values are 30-50% of normal in pituitaries of hpg male mice, and testicular LH receptors only 8% of normal (160.4 +/- 17.6 and 2013 +/- 208.1 fmol/testis respectively). In male hpg mice bearing fetal preoptic area (POA) hypothalamic implants for 10 days there was no change in pituitary GnRH receptors, pituitary gonadotrophin content, or seminal vesicle weight. However, testicular weights and LH receptors were doubled in 4/10 mice and 2 had increased serum FSH levels. Between 26 and 40 days after implantation pituitary GnRH receptors and pituitary LH increased to normal male levels, although at 40 days serum and pituitary FSH concentrations had reached only 50% of normal values. Testicular and seminal vesicle weights increased more than 10-fold by 40 days after implantation and LH receptors to 70% of normal. In hpg female mice bearing hypothalamic implants for 30-256 days pituitary gonadotrophin concentrations were normal, even though GnRH receptors reached only 60% of normal values (6.18 +/- 0.4 and 9.8 +/- 0.4 fmol/pituitary respectively). Serum FSH was substantially increased from values of less than 30 ng/ml in hpg mice to within the normal female range in hypothalamic implant recipients. Ovarian and uterine weights increased after hypothalamic grafting from only 4-5% to over 74% of normal values. LH receptors increased from 6.5 +/- 1.3 fmol/ovary for hpg mice to 566.9 +/- 39.2 fmol/ovary for implant recipients. Vaginal opening occurred about 23 days after implantation and these animals displayed prolonged periods of oestrus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of estrogen, pituitary gonadotropins and prolactin on immunohistochemical localization of inhibin subunits in the ovary of hypophysectomized female rats.

Effects of estrogen, pituitary gonadotropins and prolactin on immunohistochemical localization of alpha- and beta A-subunits in the ovaries of hypophysectomized female rats were investigated. Hypophysectomy resulted in disappearance of immunoreactive inhibin subunits in the ovary. Administration of DES, FSH and LH to hypophysectomized rats provoked growth of follicles, and resulted in positive immunostaining for inhibin subunits in the granulosa cells. In contrast to follicle-stimulating hormone (FSH) and luteinizing hormone (LH), prolactin administration failed to demonstrate positive staining for inhibin subunits in the ovary. The present in vivo results suggest that several hormones which are known to stimulate granulosa cell growth and maturation, such as estrogen, FSH and LH, enhance inhibin subunit production by the ovary. The morphologic aspect of inhibin subunit production by the ovary in response to several hormones has been demonstrated in the present study.     

Gonadotropin receptor of a mouse luteoma: interactions with luteinizing hormone (LH) and its and subunits

A radioligand-receptor system for luteinizing hormone (LH), USING transplantable mouse luteoma, was used to investigate the interactions of LH, other peptide hormones, and LH subunits. Since tumor size decreased as did production of androgenic hormones following hypophysectomy, the luteoma is believed to have been dependent on pituitary tropic hormones; posthypophysectomy histologic changes supported this conclusion. An homogenate was prepared from 1-4 gm luteomas, which had been borne by mice for 4-10 months. Ovine LH, bovine LH, and human chorionic gonadotrophin reduced the binding of iodine-125 human luteinizing hormone (125-I-hLH). Growth hormone, adrenocorticotrophic hormone, and prolactin had no capacity to interfere with binding of 125-I-hLH. Though follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH) reduced the binding somewhat, the reductions were consistent with the known presence of contaminating amounts of LH in the FSH and TSH. The accumulated results of a number of experiments suggest that binding to the luteoma LH receptor requires a particular polypeptide structural conformation, one found in the native hormone but found in neither alpha nor beta subunit alone.     

Pituitary response to LHRH, LH pulsatility and plasma melatonin and prolactin changes in ewe lambs treated with melatonin implants to delay puberty

Prepubertal ewe lambs were treated with empty or filled melatonin implants. The implants were placed s.c. at birth and pituitary responsiveness to various doses of LHRH, LH/FSH pulsatility and prolactin and melatonin secretion were examined at 10, 19, 28, 36 and 45 weeks of age. Control animals (N = 10) showed no consistent alteration in pituitary responsiveness to LHRH during development. Ewes treated with melatonin (N = 10) had puberty onset delayed by 4 weeks (P less than 0.03) but no effect of melatonin on LH or FSH response to LHRH injection was observed at any stage of development. In the control and melatonin-treated ewe lambs the responses to LHRH injection were lower during darkness than during the day at all stages of development. No consistent differences in LH or FSH pulsatility were observed between treatment groups or during development. Prolactin concentrations, however, failed to decrease at the time of puberty (autumn) in the melatonin-treated group. Melatonin-treated ewe lambs maintained normal rhythmic melatonin production which was superimposed on a higher basal concentration and showed the same increase in melatonin output with age as the control ewes. These results indicate that the delayed puberty caused by melatonin implants is not due to decreased pituitary responsiveness to LHRH or to dramatic changes in basal LH or FSH secretion.     

Plasma and pituitary concentrations of LH, FSH and prolactin in aged female C57BL/6 mice

Plasma and pituitary concentrations of LH, FSH and prolactin were determined by radioimmunoassay in 2-month-old (young) and 16-20-month-old (old) C56BL/6 mice. There were no statistical differences in hormonal levels between aged females in oestrus (those exhibiting a copulatory plug) and those in constant dioestrus. In the old females plasma levels of LH (P < 0.002) and FSH (P < 0.001) were significantly elevated, while levels of prolactin (P < 0.001) were significantly depressed when compared with those from young animals. Pituitary homogenates from old females also contained more gonadotrophins (P < 0.001) and less prolactin (P < 0.001) than those of the young females. A radioreceptor assay utilizing a plasma membrane of luteinized rat or mouse ovaries indicated that LH from 2-month-old animals bound better to ovarian receptors (P < 0.05) than did LH from old mice, although radioimmunoassay of the same samples gave higher (P < 0.01) plasma LH levels for the old mice. Since the radioreceptor assay is considered to be a more sensitive test for biologically active LH, the results from these two types of assays suggest that there may be an alteration in the mouse LH molecule with age.     

Patterns of plasma progesterone, androgen and oestrogen concentrations and in-vitro ovarian steroidogenesis during embryonic diapause and implantation in the mink (Mustela vison)

Peripheral plasma progesterone concentrations exhibited an increase 10 days before implantation, coinciding with the resumption of blastocyst growth and with a decrease in plasma androgen values (DHA, androstenedione, testosterone). No definite pattern of oestrone was observed and oestradiol concentrations remained undetectable. The production of steroids by dispersed luteal cells showed that the growth of the corpora lutea paralleled that of blastocysts and resulted in hypertrophy followed by hyperplasia of the luteal cell. The production of progesterone in the medium increased with blastocyst size up to implantation; it was enhanced by mink charcoal-treated serum, but prolactin, LH, FSH or a combination of these hormones did not affect the progesterone production, whatever the stage of diapause. DHA and androstenedione secretion increased in the two last stages of blastocyst growth and was enhanced by LH. The conversion of androstenedione and testosterone into oestrone and oestradiol was observed at all stages of embryonic diapause, indicating that corpora lutea contain aromatase activity even at an early stage. The secretion of oestrone was higher than that of oestradiol. The non-luteal tissue contributed up to 50% of the steroid production; while progesterone and androgen production remained constant, that of oestradiol decreased at the end of the delay period. These results indicated a change in the size and the secretory capacity of the luteal cell related to blastocyst development and implantation. Although progesterone was the main product of the corpora lutea, androgens and oestrogens were also secreted.     

Evidence for a pituitary site of gonadal steroid stimulation of GnRH receptors in female mice

Treatment of GnRH-deficient (hpg) female mice with oestradiol-17 beta (E2) for 7 days increased GnRH receptors from 4.1 +/- 0.4 fmol/pituitary (control) to 7.2 +/- 0.7 fmol/pituitary (GnRH-treated), and consistently increased pituitary FSH content. Treatment of hpg female mice with E2 plus progesterone (P) for 14 days stimulated GnRH receptors more than did E2 alone, although values still remained lower than those of normal intact female mice. In contrast, GnRH treatment of intact hpg female mice alone, or combined with E2 + P, increased GnRH receptors to values similar to those of intact normal female mice. In contrast, the receptor rise after GnRH treatment alone of ovariectomized hpg mice was significantly less than in intact hpg mice similarly treated. However, the combination of GnRH + E2 + P treatment of ovariectomized hpg mice increased GnRH receptors to normal intact female values, indicating the synergistic actions of these hormones on GnRH receptor up-regulation at the pituitary. Oestradiol treatment of ovariectomized normal female mice prevented the receptor fall after ovariectomy, and when combined with exogenous GnRH further increased receptors to values identical to those of intact female mice receiving GnRH alone. Ovariectomy of hpg mice had no effect on GnRH receptor, serum or pituitary LH and FSH values. There was no change in serum LH concentration after GnRH treatment of hpg female mice, but serum FSH increased and this was accentuated by ovariectomy, indicating that in intact mice an ovarian factor(s) normally inhibits GnRH-stimulated FSH release. This factor did not appear to be an ovarian steroid since serum FSH was not suppressed in intact or ovariectomized GnRH-treated hpg mice concurrently receiving E2 + P treatment. These results suggest that: (1) gonadal steroids alone have a major direct stimulatory action on the pituitary to increase GnRH receptors; (2) the oestrogen-induced increase in GnRH receptors is enhanced in the presence of GnRH; (3) steroids exert inhibitory feedback on gonadotrophin secretion that is mediated at some cellular regulatory locus other than the GnRH-receptor complex.     

Analysis of androgen action on pituitary gonadotropin and prolactin secretion in ewes

To study the role of androgens in the control of gonadotropin and prolactin secretion in ther ewe, we have characterized androgen receptors in pituitary cytosol, and investigated the effect of androgens on pituitary hormone release in vivo and in vitro. High affinity, low capacity receptors, with an affinity for methyltrienolone (R1881) greater than 5 alpha-dihydrotestosterone (5 alpha-DHT) greater than testosterone (T) much greater than androstenedione (A4), estradiol-17 beta (E2) and progesterone (P), were identified in pituitary cytosol. Addition of 1 nM 5 alpha-DHT, but not A4, inhibited luteinizing hormone (LH) release from pituitary cells in vitro, induced by 10(10) to 10(-7) M luteinizing hormone releasing hormone (LHRH). The release of follicle-stimulating hormone (FSH) with 10(-9) M LHRH was inhibited when cells were incubated with 1 nM 5 alpha-DHT. 5 alpha-DHT had no effect when higher or lower doses of LHRH were used. In ovariectomized ewes, neither an i.v. injection of 1 mg, nor intracarotid injections of up to 1 mg, 5 alpha-DHT affected plasma LH, FSH or prolactin levels, despite dose-related increases in plasma 5 alpha-DHT levels. Daily or twice daily i.m. injections of 5 mg 5 alpha-DHT in oil did not affect LH or FSH levels, but daily injections of 20 mg significantly reduced plasma LH levels within 4 days and plasma FSH levels within 6 days. Thus, despite the presence of androgen receptors in the ewe pituitary, we conclude that androgens per se are of minimal importance in the regulation of pituitary LH, FSH and prolactin secretion in the ewe. The low binding affinity of A4 and the lack of its effect on hormone secretion in vitro suggests that A4 may act as an estrogen precursor rather than an androgenic hormone. The function of the pituitary androgen receptor remains to be established.     

Progesterone does not affect the amount of mRNA for gonadotropins in the anterior pituitary gland of ovariectomized ewes

To evaluate the effect of progesterone on the synthesis and secretion of gonadotropins, ovariectomized ewes either were treated with progesterone (n = 5) for 3 wk or served as controls (n = 5) during the anestrous season. After treatment for 3 wk, blood samples were collected from progesterone-treated and ovariectomized ewes. After collection of blood samples, hypothalamic and hypophyseal tissues were collected from all ewes. Half of each pituitary was used to determine the content of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and the number of receptors for gonadotropin-releasing hormone (GnRH). The amounts of mRNA for LH beta subunit, FSH beta subunit, alpha subunit, growth hormone, and prolactin were measured in the other half of each pituitary. Treatment with progesterone reduced mean serum concentrations of LH (p less than 0.001) but ot FSH (p greater than 0.05). Further, progesterone decreased (p less than 0.05) the total number of pulses of LH. We were unable to detect pulsatile release of FSH. Hypothalamic content of GnRH, number of receptors for GnRH, pituitary content of gonadotropins and mRNA for LH beta subunit, FSH beta subunit, alpha subunit, growth hormone, and prolactin were not affected (p greater than 0.05) by treatment with progesterone. Thus, after treatment with progesterone, serum concentrations of LH (but not FSH) are decreased. This effect, however, is not due to a decrease in the steady-state amount of mRNA for LH beta or alpha subunits.     

Effect of bromocriptine on the hypothalamo-pituitary function in adult male rats.

Daily oral administration of bromocriptine (50 μg/kg) to adult male rats, suppressed serum prolactin levels. The pituitary prolactin levels remained unaltered. Serum FSH levels as well as pituitary FSH levels showed no significant change as compared to the controls. Serum LH levels were significantly decreased in spite of the high pituitary LH levels, in bromocriptine treated rats. In the drug treated rats, $\text{in vitro}$ sensitivity of the pituitary to the exogenous LH-RH was not altered; whereas hypothalamic LH-RH content was considerably lowered. These observations suggest the possible effect of bromocriptine on the synthesis of LH-RH in the hypothalamus which leads to the accumulation of LH in the pituitary and decline of serum LH.     

Preliminary observations on the existing relationship between plasma levels of prolactin and hypophyseal reserves of FSH and LH in the male

PRL plasma levels and FSH and LH pituitary reserve were tested in ten apparently healthy male subjects. A good correlation was found between PRL on one hand and FSH plasma levels (p less than 0,05), LH plasma levels (p less than 0,01) and FSH pituitary reserve (p less than 0,01) on the other hand. This seems to support the current hypothesis that prolactin may cause a progressive clinically latent impairment in the spermatogenetic function of the testis. Further evidence is needed.     

Evidence that gonadotropin-releasing hormone (GnRH) II stimulates luteinizing hormone and follicle-stimulating hormone secretion from monkey pituitary cultures by activating the GnRH I receptor

Mammalian gonadotropin-releasing hormone (GnRH) I is the neuropeptide that regulates reproduction. In recent years, a second isoform of GnRH, GnRH II, and its highly selective type II GnRH receptor were cloned and identified in monkey brain, but its physiological function remains unknown. We sought to determine whether GnRH II stimulates LH and FSH secretion by activating specific receptors in primary pituitary cultures from male monkeys. Dispersed pituitary cells were maintained in steroid-depleted media and stimulated with GnRH I and/or GnRH II for 6 h. Cells were also treated with Antide (Bachem, King of Prussia, PA), a GnRH I antagonist, to block gonadotropin secretion. In monkey as well as rat pituitary cultures, GnRH II was a less effective stimulator of LH and FSH secretion than was GnRH I. In both cell preparations, Antide completely blocked LH and FSH release provoked by GnRH II as well as GnRH I. Furthermore, the combination of GnRH I and GnRH II was no more effective than either agonist alone. These results indicate that GnRH II stimulates FSH and LH secretion, but they also imply that this action occurs through the GnRH I receptor. The GnRH II receptors may have a unique function in the monkey brain and pituitary other than regulation of gonadotropin secretion.     

Partial purification and characterization of rhinoceros gonadotropins, growth hormone, and prolactin: comparison with the horse and sheep

The rhinoceros is an endangered species related to the horse family. Little is known of its reproductive endocrinology. The objectives of this study were to partially purify rhinoceros pituitary hormones, determine which assays could be used for their assessment, and to ascertain whether rhinoceros LH possesses the intrinsic FSH activity of equine LH. A single pituitary each from a White (1.3 g) and a Black (1.2 g) Rhinoceros was homogenized and extracted (pH 9.5), then subjected to pH and salt fractionation, and ion-exchange chromatography (DEAE and Sephadex SP-C50) to yield partially purified fractions of LH, FSH, growth hormone (GH), and prolactin (PRL). LH was readily measured by a rat Leydig cell assay (0.1-1% x equine LH) and an RIA using a monoclonal antibody to bovine LH (6-11% x equine LH). FSH activity detected in the LH by either an FSH RIA or a calf testis radioreceptor assay (RRA) was extremely low. No FSH activity could be detected in the White Rhinoceros pituitary "FSH" fraction, but was readily detected in the Black Rhinoceros fraction (RIA: 0.2% x equine FSH: RRA: 0.8% x equine FSH). The presence of GH and PRL was determined by SDS-PAGE and Western blots. Results showed a single immunoreactive GH band and multiple immunoreactive PRL bands. Adsorption with Concanavalin A-Sepharose indicated that some of the PRL bands are glycosylated.     

Chronobiological analysis of changes in circadian rhythm of anterior pituitary hormones level during glycemia normalization by means of biostator

We compared the circadian rhythms of anterior pituitary hormones in 15 patients with noncompensated insulin-dependent diabetes on first and second day treatment with Biostator. The rhythm was evaluated by means of a least squares analysis and presented as the circle of cosinors. In noncompensated diabetes the TSH and prolactin rhythm was maintained, whereas other hormones of the anterior pituitary showed no significant rhythm. In the course of one-day normalization of glycemia by means of Biostator the TSH and prolactin rhythm was maintained, whereas the circadian rhythm of growth hormone and ACTH levels appeared with acrophase at 18.47 and 19.59 hour, respectively. The LH rhythm did not exist, whereas the FSH rhythm was dubious. One may assume that noncompensated diabetes results in the impairment of certain pituitary hormonal rhythms and these disturbances are reversible after restoring of normoglycemia.     

Effects of naloxone,morphine and methionine enkephalin on serum prolactin,luteinizing hormone,follicle stimulating hormone,thyroid stimulating hormone and growth hormone

The response of 5 anterior pituitary hormones to single injections of naloxone, morphine and metenkephalin administration was measured in male rats. Morphine and met-enkephalin significantly increased serum prolactin and GH concentrations, and significantly decreased serum LH and TSH concentrations. Naloxone reduced serum prolactin and GH concentrations, increased serum LH and FSH, but had little effect on serum TSH concentrations. Concurrent injections of naloxone with morphine or met-enkephalin reduced the response to each of the drugs given separtely. These results suggest that endogenous morphinomimetic substances may participate in regulating secretion of anterior pituitary hormones.     

Interactions of prostaglandin E1 (PGE1) and LRH on anterior pituitary function

Numerous biochemical pathways influence the synthesis and release of anterior pituitary hormones. Releasing factors extracted from the hypothalamus and prostaglandins (PGs) appear to alter a common biochemical activity, adenyl cyclase, in pituitary cells. Luteinizing hormone releasing hormone (LRH), prostaglandin (PGE₁), 7 oxa-13-prostynoic acid and cycloheximide were tested for individual and interacting effects on the in vitro release of FSH, LH and prolactin from hemipituitaries of 15 day old female rats. LRH (10 ng/ml) consistently released both LH and FSH in all in vitro experiments and inhibited prolactin release in 1 of 2 experiments. Lower concentrations (5 and 1 ng/ml) also stimulated LH and FSH release but did not influence prolactin release. Concurrent depletion of stored LH and FSH in the gland was observed. PGE₁ in a 6.5 hour incubation increased the storage of LH within the gland in the absence of LRH. In a 1.5 hour incubation in the presence of LRH, storage of LH was also increased. PGE₁ had no effect on LH and FSH release; however, in 1 of 2 experiments it stimulated prolactin release in the absence of LRH. Prostynoic acid stimulated LH and FSH release but did not synergize with LRH action in the same tissue. Cycloheximide did not affect LH release during the first 30 minutes of incubation; however, the release during the subsequent 1 hour was significantly inhibited. Similar tissue also exposed to cycloheximide was still responsive to LRH during the latter 1 hour incubation period. Cycloheximide had no effect on prolactin storage and release from the same tissue.     

Effects of hypothalamic neurohormones on prolactin release from pituitary allografts in the hamster

Recent reports indicate that luteinizing hormone-releasing hormone (LHRH) releases prolactin (PRL) under some circumstances. We examined the chronic effects of LHRH, growth hormone-releasing hormone (GHRH), and corticotrophin-releasing hormone (CRH) on the release of PRL, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) by pituitary allografts in hypophysectomized, orchidectomized hamsters. Entire pituitary glands removed from 7-week-old-male Golden Syrian hamsters were placed under the renal capsule of hypophysectomized, orchidectomized 12-week-old hamsters. Beginning 6 days postgrafting, hamsters were injected subcutaneously twice daily with 1 microgram LHRH, 4 micrograms GHRH, or 4 micrograms CRH in 100 microliter of vehicle for 16 days. Six hosts from each of the four groups were decapitated on Day 17, 16 hr after the last injection. Prolactin, LH, and FSH were measured in serum collected from the trunk blood. Treatment with LHRH significantly elevated serum PRL levels above those measured in the other three groups, which were all similar to one another. Serum LH levels in hosts treated with vehicle were elevated above those measured in the other three groups. Serum FSH levels in hosts treated with LHRH were greater than FSH levels in any of the other three groups. These results indicate that chronic treatment with LHRH can stimulate PRL and FSH release by ectopic pituitary cells in the hamster.     

Effects of hyperprolactinemia on the control of luteinizing hormone and follicle-stimulating hormone secretion in the male rat

Experiments were conducted to determine the effects of acute hyperprolactinemia (hyperPRL) on the control of luteinizing hormone and follicle-stimulating hormone secretion in male rats. Exposure to elevated levels of prolactin from the time of castration (1 mg ovine prolactin 2 X daily) greatly attenuated the post-castration rise in LH observed 3 days after castration. By 7 days after castration, LH concentrations in the prolactin-treated animals approached the levels observed in control animals. HyperPRL had no effect on the postcastration rise in FSH. Pituitary responsiveness to gonadotropin hormone-releasing hormone (GnRH), as assessed by LH responses to an i.v. bolus of 25 ng GnRH, was only minimally effected by hperPRL at 3 and 7 days postcastration. LH responses were similar at all time points after GnRH in control and prolactin-treated animals, except for the peak LH responses, which were significantly smaller in the prolactin-treated animals. The effects of hyperPRL were examined further by exposing hemipituitaries in vitro from male rats to 6-min pulses of GnRH (5 ng/ml) every 30 min for 4 h. HyperPRL had no effect on basal LH release in vitro, on GnRH-stimulated LH release, or on pituitary LH concentrations in hemipituitaries from animals that were intact, 3 days postcastration, or 7 days postcastration. However, net GnRH-stimulated release of FSH was significantly higher by pituitaries from hyperprolactinemic, castrated males. To assess indirectly the effects of hyperPRL on GnRH release, males were subjected to electrical stimulation of the arcuate nucleus/median eminence (ARC/ME) 3 days postcastration. The presence of elevated levels of prolactin not only suppressed basal LH secretion but reduced the LH responses to electrical stimulation by 50% when compared to the LH responses in control castrated males. These results suggest that acute hyperPRL suppresses LH secretion but not FSH secretion. Although pituitary responsiveness is somewhat attenuated in hyperprolactinemic males, as assessed in vivo, it is normal when pituitaries are exposed to adequate amounts of GnRH in vitro. Thus, the effects of hyperPRL on pituitary responsiveness appear to be minimal, especially if the pituitary is exposed to an adequate GnRH stimulus. The suppression of basal LH secretion in vivo most likely reflects inadequate endogenous GnRH secretion. The greatly reduced LH responses after electrical stimulation in hyperprolactinemic males exposed to prolactin suggest further that hyperPRL suppresses GnRH secretion.     

Prostacyclin (PGI2) effects on anterior pituitary hormones in the rat in vivo.

Intravenous injection of 600 microgram PGE2 or PGI2 significantly increased serum LH and prolactin levels in estradiol treated ovariectomized rats. There was no effect on serum FSH concentration. PGE2 and PGI2 stimulated LH release in a non-dose dependent manner, while prolactin levels were positively correlated with the dose administered following PGI2 treatment. 6-keto-PGF1 alpha at a comparable dose had no effect on pituitary hormone levels. Subcutaneous administration of 1 mg/kg or 60 mg/kg PGI2 for seven days significantly depressed serum LH level both in male and female rats. These doses had no effect on serum FSH or prolactin levels.     

Twinning and the changing pattern of breast‐feeding: A possible relationship in a small rural population

Abstract

Direct and indirect observations suggest that pituitary hormones, namely FSH, LH, GnRH, and GH, are involved in the mechanism of twinning. We hypothesized that prolactin might be involved as well. It is well known that lactation prolactin induces a gonadotropin‐mediated amenorrhea. Otherwise, weaning results in low serum prolactin and high FSH and LH secretion leading to the resumption of ovulation. Thus, variations in serum prolactin in women carriers of a twin gene may have a bearing on their gonadotropin‐mediated twinning liability. The hypothesis was tested using data from a small rural population characterized by a shift from total breast‐feeding to partial breast‐feeding with an early introduction of solid foods to infants.