Gonadotropin-releasing hormone neuron requirements for puberty, ovulation, and fertility

Abstract

The absolute requirement for reproduction implies that the hypothalamo-pituitary-gonadal axis, controlling fertility, is an evolutionary robust mechanism. The GnRH neurons of the hypothalamus represent the key cell type within the body dictating fertility. However, the level of functional redundancy within the GnRH neuron population is unknown. As a result of a fortuitous transgene insertion event, GNR23 mice exhibit a marked allele-dependent reduction in GnRH neuron number within their brain. Wild-type mice have approximately 600 GnRH neurons, compared with approximately 200 (34%) and approximately 70 (12%) in GNR23(+/-) and GNR23(-/-) mice, respectively. Using these mice, we examined the minimal GnRH neuron requirements for fertility. Male GNR23(-/-) mice exhibited normal fertility. In contrast, female GNR23(-/-) mice were markedly subfertile, failing to produce normal litters, have estrous cycles, or ovulate. The failure of ovulation resulted from an inability of the few existing GnRH neurons to generate the LH surge. This was not the case, however, for the first cycle at puberty that appeared normal. Together, these observations demonstrate that 12% of the GnRH neuron population is sufficient for pulsatile gonadotropin secretion and puberty onset, whereas between 12 and 34% are required for cyclical control in adult female mice. This indicates that substantial redundancy exists within the GnRH neuronal population and suggests that the great majority of GnRH neurons must be dysfunctional before fertility is affected.

Figure 1

Allele-dependent reduction in GnRH neuron numbers in the brains of GNR23 homozygous (-/-) and hemizygous (-/-) female mice. A-F, coronal brain sections showing GnRH immunoreactivity in the rostral preoptic area (rPOA) (A-C) and median eminence (D-F). Note that the numbers of GnRH cell bodies and their projections to the median eminence are diminished in a step-wise manner according to genotype. G, mean + SEM numbers of GnRH neurons per 30μm-thick brain section in the medial septum, rPOA and anterior hypothalamic area (AHA). The distribution of GnRH neurons (black dots) at these three levels is given in schematic coronal brain sections at the bottom. * p<0.01 (ANOVA with post-hoc Tukeys) compared with wild-type.

Figure 2

Puberty onset in GNR23 female mice. The mean (+SEM) day of vaginal opening and first estrus is given for each of the three genotypes. Wild-type (wt, n=11); hemizygous (+/-, n=23); homozygous (-/-, n=6), * p<0.05 (ANOVA with post-hoc Tukeys).

Figure 3

Reproductive phenotype of GNR23 male mice. Histograms show mean (+SEM) (A) testis weight, (B) gonadotropin secretion and (C) numbers of litters born to GNR23 male mice/3 months and numbers of pups/litter for wild-type (wt), hemizygous (+/-) and homozygous (-/-) GNR23 mice. * p<0.05, ** p<0.01 (ANOVA with post-hoc Tukeys).

Figure 4

Fertility of GNR23 mice. A, Schematic representation of females’ reproductive history following introduction of a wild-type male mouse (beginning of mating, arrow = day 0). Each line represents time across 140 days for an individual female mouse with arrowheads indicating the day each litter was born. Data for 7 wild-type, 6 hemizygous (+/-) and 14 homozygous (-/-) females are given. B, Mean (+SEM) number of litters for each genotype. C, Mean (+SEM) number of pups born/litter for the three genotypes. D, Mean (+SEM) number of days of the estrous cycle for wild-type (wt), and GNR23 female mice. Note that -/- data only include those GNR23 female mice that showed evidence of a cycle; most were in persistent estrus. *** p<0.01, * p<0.05 (ANOVA with post-hoc Tukeys).

Figure 5

Histological sections of wt (A, B) and hmz (C, D) ovaries. Normal stromal tissue with vascular elements is present throughout the wt samples (arrows). Extensive areas of flocculent cellular material is seen in the stroma of the hmz specimen (arrowheads). Antral follicles are indicated in A and C (asterisks). Scale bars represent 45μm in A and C and 15μm in B and D.

Figure 6

LH surge in GNR23 mice. A, Profile of LH surge in OVX mice of all three genotypes treated with estrogen and progesterone. Note that the LH surge in hemizygous (+/-) mice (* p<0.05) is markedly flattened in profile whereas evaluation of LH levels at the time of peak LH secretion (18:00h) reveals an absence of the LH surge in homozygous (-/-) GNR23 females. Bar at bottom indicates light schedule. B, Pituitary response to exogenous GnRH. Mean (+SEM) LH levels before (pre-) and 10 min after (post-) 200ng/kg GnRH s.c. to wild-type (black bars) and homozygous GNR23 (open bars) female mice. * p<0.05. C, Dual-label immunocytochemistry showing a GnRH neuron expressing c-Fos (black nuclear staining) in the rostral preoptic area (rPOA) of a hmz GNR23 mouse. D, Mean (+SEM) number of medial septal (MS) and rPOA GnRH neurons/coronal section expressing c-Fos at 18:00h in the three genotypes. E, Mean (+SEM) percentage of rPOA GnRH neurons expressing c-Fos at 18:00h. Scale bar in C represents 50μm.