Male Reproductive Function Is Not Affected in Prolactin Receptor ...

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Endocrinology 144(9):3779 –3782 Copyright © 2003 by The Endocrine Society doi: 10.1210/en.2003-0409

Male Reproductive Function Is Not Affected in Prolactin Receptor-Deficient Mice NADINE BINART, NATHALIE MELAINE, CHARLES PINEAU, HENRI KERCRET, ´ , PAUL A. KELLY, AND BERNARD JE ´ GOU ANNE MARIE TOUZALIN, PRUNE IMBERT-BOLLORE Hormone Targets, Institut National de la Sante´ et de la Recherche Me´dicale Unite´ 584 (N.B., P.I-B., P.A.K.), Faculte´ de Me´decine Necker-Enfants Malades, 75015 Paris, France; and Groupe d’Etude de la Reproduction chez le Maˆle-Institut National de la Sante´ et de la Recherche Me´dicale Unite´ 435 (N.M., C.P., H.K., A.M.T., B.J.), Universite´ de Rennes 1, 35042 Rennes Cedex, Bretagne, France Mice with a targeted disruption of the prolactin (PRL) receptor gene were used to study the physiological role of PRL in the control of the male reproductive function. Fertility parameters as well as body and reproductive organ weights (epididymis and testes) were unaffected in PRL receptor knockout mice. Testicular histology and sperm reserves were also normal. Compared with wild-type animals, knockout mice

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HEREAS PROLACTIN (PRL) has long been known to be the hormone responsible for mammary gland development and lactation in females, its role in the male has puzzled investigators ever since it has been shown to be present in the anterior pituitary. Initially, no clear function could be ascribed to PRL in male mammals, including humans (1, 2). However, more recent data have suggested that, generally, this hormone positively modulates several aspects of testicular function. Thus, PRL has been presented as being involved in the maintenance of cellular morphology (3) and in the up-regulation of LH receptor number on Leydig cells (4, 5). Along with LH, it has also been proposed to be implicated in the stimulation of steroidogenesis and androgen production (5–7), whereas, in contrast, it could be involved in the inhibition of aromatase activity (8). In vitro, PRL has been shown to increase FSH receptor number in Sertoli cells (9). It has also been suggested that PRL is involved in the rate of spermatocyte-spermatid conversion (3). Moreover, several in vitro effects on spermatozoa have been reported: a rise in calcium binding and/or transport of ejaculated and epididymal spermatozoa (10), an increase in energy metabolism (11), a maintenance of mobility and attachment to the oocyte (12), and a reduction in the time required to achieve capacitation (12). PRL also has metabolic effects on sex accessory organs (13–15). The effects on prostate include increased levels of androgen receptors (16, 17), involvement in estrogeninduced inflammation (18), increased epithelial secretory function (19, 20), and augmented energy metabolism. Stimulation of the level of IGF-I and its receptor has been also reported in the prostate (17). In addition to these in vitro data, it has been shown that, in the mouse, congenital PRL deficiency caused by recessive mutations at the pit-1 locus (Snell dwarf; Ames dwarf) is Abbreviations: hCG, Human chorionic gonadotropin; KO, knockout; PRL, prolactin; PRLR, PRL receptor.

had no significant difference in basal plasma LH, FSH, and testosterone levels, and the weight of seminal vesicles and prostate was unaffected. Moreover, no alteration was detected in human chorionic gonadotropin-induced testosterone levels. It is concluded that the absence of PRL signaling is not detrimental to male testicular function and to fertility in the mouse. (Endocrinology 144: 3779 –3782, 2003)

associated with reduced testosterone levels, a decrease in testicular LH and PRL receptor (PRLR) number, and a severe suppression of fertility (21, 22). However, these effects may well result from a drop in circulating gonadotropin levels observed in these studies. In contrast, the model of PRL knockout (KO) mouse has allowed the demonstration that, if PRL has a physiological role in the control of LH release and in the regulation of the growth of accessory reproductive glands, it does not seem to be directly required for the maintenance of circulating testosterone and fertility (23). The development of PRLR KO mice (24) has allowed us in the present study to reinvestigate the possible involvement of PRL in testicular function. We demonstrate that none of the male reproductive tract organ parameters or functions investigated was affected in this model. Materials and Methods Animals and mating trials The animals were produced by crossing animals heterozygous for the PRLR. Mice were housed under normal laboratory conditions in a 12-h light, 12-h dark cycle (0700 –1900). The temperature was controlled (21 C), and the animals had free access to tap water and standard pelleted animal food. The progeny was classified by PCR analysis of DNA extracted from tails clipping as described previously (25). After reaching adulthood (2–3 months of age), each male was placed for 30 d in a cage with two virgin females, and then the females were checked daily for the presence of a vaginal plug. Mating behavior (frequency of mounting and copulation) was analyzed, and immediately after parturition the size of the litter was recorded. The local committee on animal care approved all animal protocols.

Human chorionic gonadotropin (hCG) test Ten PRLR KO and 10 wild-type mice were chosen at random and treated with an ip injection of hCG (Organon, Puteaux, France) in PBS, at 15 IU/animal. Controls (n ⫽ 10) were injected with PBS alone. Two hours after injection, all animals were decapitated, the blood collected, and the plasma separated and stored at ⫺20 C for hormones assays.

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Binart et al. • Brief Communications

forced by our data, which did not show any change in body weight of young KO animals (31.90 ⫾ 0.43/32.80 ⫾ 0.47 for ⫹/⫹ mice). No change was seen in testis, epididymis, seminal vesicle, or prostate weights, and testis histology was normal (Fig. 1). Moreover, sperm reserves were similar to those of controls (Fig. 2). Fertility study

Neither the frequency of mounting and copulation nor the number of observed vaginal plugs in wild-type females mated to PRLR KO or to wild-type males was changed, indicating a normal mating behavior. All PRLR KO males similar to wild-type males were fertile (100% fertile, average litter size 8.2 ⫾ 0.3, n ⫽ 22; and 8.7 ⫾ 0.2, n ⫽ 15, respectively), and they continued to produce litters of normal size as they grew older. In contrast to our previous observation suggesting a delay of male fertility (24), no change of this parameter was seen in close studies. Hormonal status

Pituitary hormones. Basal plasma FSH and LH levels were not significantly changed in PRLR KO vs. wild-type males (Table 1). Testosterone levels. Testosterone levels did not differ significantly between genotypes (Fig. 3). In agreement with these observations, the seminal vesicles and prostate weights were not affected in homozygous PRLR KO, compared with wildtype males (Fig. 1). Furthermore, the response to hCG administration was not modified in the PRLR KO animals (Fig. 3). Discussion FIG. 1. Reproductive tract organ weights and testis histology in adult wild-type male (⫹/⫹) and PRLR KO (⫺/⫺) mice. Values are the mean ⫾ SEM for 30 mice. Scale bar, 100 ␮m.

Collection of tissues

In the past, a number of studies have pointed out the possible influence of PRL in the control of various aspects of male reproductive function. However, until recently, direct effects of PRL, such as the increase in Leydig cell LH receptor number (4), the enhanced sensitivity of Leydig cells to LH

At the time of autopsy, testes, epididymis, ventral prostate, and seminal vesicles of the KO and wild-type mice were collected and weighed. One testis was immediately fixed in Bouin’s fluid for histology, whereas the other testis was stored at ⫺20 C until sperm reserves were counted as previously described (26). Blood plasma was saved for measurements of LH, FSH, and testosterone by RIAs previously validated for use in mouse plasma. The serum concentrations of LH and FSH were measured using [125I]LH, [125I]FSH, and materials obtained from the National Hormone and Pituitary Program. The lower limits of detection for these assays are 0.11 ng/ml and 1 ng for LH and FSH, respectively; values are expressed in relation to the standards. The intra- and interassay coefficients of variation were 5 and 7%, respectively. Serum testosterone levels were measured with a tritium-based RIA, with a 10 –13% interassay variation. All samples were measured in the same assay. The results were expressed as the mean ⫾ sem. The variance analyses using unpaired two-tailed Student’s t test were used for comparing the values measured between PRLR KO and wild-type animals.

Results Body and reproductive organ weights

No apparent change was seen in the health status of PRLR KO mice compared with wild-type animals. This was rein-

FIG. 2. Sperm reserves of homozygous PRLR⫺/⫺ mutants and wildtype mice. Total epididymal sperm was counted in adult ⫹/⫹ (white bar) and ⫺/⫺ (black bar) mice. Values are the mean ⫾ SEM for 12 wild-type and 16 PRL ⫺/⫺ mice.


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TABLE 1. Circulating FSH and LH levels in adult wild-type and PRLR KO male mice

FSH (ng/ml) LH (ng/ml)

⫹/⫹

⫺/⫺

24.47 ⫾ 3.79 (n ⫽ 27) 2.20 ⫾ 0.57 (n ⫽ 8)

24.75 ⫾ 6.80 (n ⫽ 27) 2.57 ⫾ 0.43 (n ⫽ 8)

Values are the mean ⫾ indicated in parentheses.

SEM;

the number of mice per group is

of the mixed genetic background used previously. Furthermore, we demonstrate that the total ablation of PRLR expression has no significant consequence on spermatogenesis as assessed by testicular histology and measurement of testicular sperm reserves, fertility in term of sexual behavior and mating outcome, and androgen status, as shown by unchanged secondary accessory organ weights and basal and hCG-induced circulating testosterone. Therefore, we conclude that PRL is not a essential player in the control of male reproductive function in the mouse. Acknowledgments Received April 1, 2003. Accepted June 30, 2003. Address all correspondence and requests for reprints to: Dr. Nadine Binart, Hormone Targets, Institut National de la Sante´ et de la Recherche Me´ dicale Unite´ 584, Faculte´ de Me´ decine Necker-Enfants Malades, 156 rue de Vaugirard, 75015 Paris, France. E-mail: [email protected]. This work was supported in part by grants from Institut National de la Sante´ et de la Recherche Me´ dicale and the Ministe`re de l’Education Nationale de la Recherche et de la Technologie (No. 1A010G).

References

FIG. 3. Plasma testosterone concentrations 2 h after injection of saline (basal) or of hCG to the wild-type littermates (⫹/⫹) and PRLR KO mice. Asterisks denote statistical differences from the respective controls (P ⬍ 0.001). NS, No significant difference when control testosterone levels were compared between ⫹/⫹ and ⫺/⫺ mice and when hCG-induced testosterone levels were compared accordingly. Values are expressed as the mean ⫾ SEM for n ⫽ 10.

stimulation (5), or the stimulation of Sertoli FSH receptor number (9), could only be ascertained in vitro. Alternatively, a few studies were undertaken in vivo, but under conditions that did not permit to discrimination between the possible direct effects of PRL and the effects that could have been mediated through alterations in the hormones affected by PRL. In fact, it is well established that PRL can alter secretion of gonadotropins by the pituitary gland with subsequent changes occurring in spermatogenesis (27). A classical example of this is that, in hyperprolactinemic men, the abnormalities of spermatogenesis observed are currently thought to be a consequence of impaired testosterone levels due to LH release defects (28). The same applies to the phenotype observed in mice affected by a congenital PRL deficiency caused by recessive mutations at the pit-1 locus (22). The recent analysis of the phenotype of genetically engineered mice null for PRL gene has represented a notable breakthrough. Thus, Steger et al. (23) have recently shown that in the absence of PRL, fertility and testosterone levels are normal. The present study using PRLR KO mice totally confirms this finding. In contrast to our previous observation suggesting a delay of male fertility (24), no change of this parameter was seen in close studies. This discrepancy is explained by the use of a larger number of animals in the present mating trials and by the fact that the present study was undertaken on pure 129/Sv genetic background, instead

1. Nicoll CS 1974 Physiological actions of prolactin. In: Knobil E, Sawyer W, eds. Handbook of physiology: endocrinology IV. Baltimore: Williams and Wilkins; vol 2:253–292 2. Bole-Feysot C, Goffin V, Edery M, Binart N, Kelly PA 1998 Prolactin and its receptor: actions, signal transduction pathways and phenotypes observed in prolactin receptor knockout mice. Endocr Rev 19:225–268 3. Nag S, Sanyal S, Ghosh KK, Biswas NM 1981 Prolactin suppression and spermatogenic developments in maturing rats. A quantitative study. Horm Res 15:72–77 4. Dombrowicz D, Sente B, Closset J, Hennen G 1992 Dose-dependent effects of human prolactin on the immature hypophysectomized rat testis. Endocrinology 130:695–700 5. Purvis K, Clausen OP, Olsen A, Haug E, Hansson V 1979 Prolactin and Leydig cell responsiveness to LH/hCG in the rat. Arch Androl 3:219 –230 6. Rubin RT, Poland RE, Tower BB 1976 Prolactin-related testosterone secretion in normal adult men. J Clin Endocrinol Metab 42:112–116 7. Gunasekar PG, Kumaran B, Govindarajulu P 1988 Prolactin and Leydig cell steroidogenic enzymes in the bonnet monkey (Macaca radiata). Int J Androl 11:53–59 8. Papadopoulos V, Drosdowsky MA, Carreau S 1986 In vitro effects of prolactin and dexamethasone on rat Leydig cell aromatase activity. Andrologia 18:79 – 83 9. Guillaumot P, Tabone E, Benahmed M 1996 Sertoli cells as potential targets of prolactin action in the testis. Mol Cell Endocrinol 122:199 –206 10. Reyes A, Parra A, Chavarria ME, Goicoechea B, Rosado A 1979 Effect of prolactin on the calcium binding and/or transport of ejaculated and epididymal human spermatozoa. Fertil Steril 31:669 – 672 11. Rui H, Lebrun JJ, Kirken RA, Kelly PA, Farrar WL 1994 Jak2 activation and cell proliferation induced by antibody-mediated prolactin receptor dimerization. Endocrinology 135:1299 –1306 12. Fukuda A, Mori C, Hashimoto H, Noda Y, Mori T, Hoshino K 1989 Effects of prolactin during preincubation of mouse spermatozoa on fertilizing capacity in vitro. J In Vitro Fert Embryo Transf 6:92–97 13. Reddy YD, Reddy KV, Govindappa S 1985 Effect of prolactin and bromocriptine administration on epididymal function: a biochemical study in rats. Indian J Physiol Pharmacol 29:234 –238 14. Gautam R, Pereira BM 1992 The effect of ovine prolactin on the epididymal sialic acid concentration in male rats. Clin Exp Pharmacol Physiol 19:495–501 15. Ray B, Gautam R, Gaur M, Srivastava N, Pereira BM 1994 Impact of prolactin on epididymal lipid profile in castrated rats. Indian J Exp Biol 32:299 –303 16. Prins GS 1987 Prolactin influence on cytosol and nuclear androgen receptors in the ventral, dorsal, and lateral lobes of the rat prostate. Endocrinology 120:1457–1464 17. Reiter E, Bonnet P, Sente B, Dombrowicz D, de Leval J, Closset J, Hennen G 1992 Growth hormone and prolactin stimulate androgen receptor, insulinlike growth factor-I (IGF-I) and IGF-I receptor levels in the prostate of immature rats. Mol Cell Endocrinol 88:77– 87 18. Tangbanluekal L, Robinette CL 1993 Prolactin mediates estradiol-induced inflammation in the lateral prostate of Wistar rats. Endocrinology 132: 2407–2416

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19. Tam CC, Wong YC, Tang F 1992 Ultrastructural and cytochemical studies of the effects of prolactin on the lateral prostate and the seminal vesicle of the castrated guinea pig. Cell Tissue Res 270:105–112 20. Edwards WD, Thomas JA 1980 Morphologic and metabolic characteristics of ventral, lateral, dorsal and anterior prostate transplants in rats, Effect of testosterone and/or prolactin. Horm Res 13:28 –39 21. Bartke A 1966 Influence of prolactin on male fertility in dwarf mice. J Endocrinol 35:419 – 420 22. Bartke A, Goldman BD, Bex F, Dalterio S 1977 Effects of prolactin (PRL) on pituitary and testicular function in mice with hereditary PRL deficiency. Endocrinology 101:1760 –1766 23. Steger RW, Chandrashekar V, Zhao W, Bartke A, Horseman ND 1998 Neuroendocrine and reproductive functions in male mice with targeted disruption of the prolactin gene. Endocrinology 139:3691–3695 24. Ormandy CJ, Camus A, Barra J, Damotte D, Lucas BK, Buteau H, Edery M,


25.

26. 27.

28.

Brousse N, Babinet C, Binart N, Kelly PA 1997 Null mutation of the prolactin receptor gene produces multiple reproductive defects in the mouse. Genes Dev 11:167–178 Binart N, Helloco C, Ormandy CJ, Barra J, Clement-Lacroix P, Baran N, Kelly PA 2000 Rescue of preimplantatory egg development and embryo implantation in prolactin receptor-deficient mice after progesterone administration. Endocrinology 141:2691–2697 Robb GW, Amann RP, Killian GJ 1978 Daily sperm production and epididymal sperm reserves of pubertal and adult rats. J Reprod Fertil 54:103–107 Behre HM, Nieschlag E, Meschede D, Partsch CJ 1997 Diseases of the hypothalamus and the pituitary gland. In: Nieschlag E, Behre HM, eds. Andrology (male reproductive health and dysfunction). New York: Springer; Chap 7:115–129 Micic S, Dotlic R, Ilic V, Genbacev O 1985 Hormone profile in hyperprolactinemic infertile men. Arch Androl 15:123–128