Roles of Stra8 and Tcerg1l in retinoic acid induced spermatogonial differentiation in mouse†.

Retinoic acid (RA) induces spermatogonial differentiation, but the mechanism by which it operates remains largely unknown. We developed a germ cell culture assay system to study genes involved in spermatogonial differentiation triggered by RA. Stimulated by RA 8 (Stra8), a RA-inducible gene, is indispensable for meiosis initiation, and its deletion results in a complete block of spermatogenesis at the pre-leptotene/zygotene stage. To interrogate the role of Stra8 in RA mediated differentiation of spermatogonia, we derived germ cell cultures from the neonatal testis of both wild type and Stra8 knock-out mice. We provide the first evidence that Stra8 plays a crucial role in modulating the responsiveness of undifferentiated spermatogonia to RA and facilitates transition to a differentiated state. Stra8-mediated differentiation is achieved through the downregulation of a large portfolio of genes and pathways, most notably including genes involved in the spermatogonial stem cell self-renewal process. We also report here for the first time the role of transcription elongation regulator-1 like (Tcerg1l) as a downstream effector of RA-induced spermatogonial differentiation.

Regulators of the protein phosphatase PP1γ2, PPP1R2, PPP1R7, and PPP1R11 are involved in epididymal sperm maturation.

The serine/threonine protein phosphatase 1 (PP1) inhibitors PPP1R2, PPP1R7, and PPP1R11 are evolutionarily ancient and highly conserved proteins. Four PP1 isoforms, PP1α, PP1ß, PP1γ1, and PP1γ2, exist; three of them except PP1γ2 are ubiquitous. The fact that PP1γ2 isoform is present only in mammalian testis and sperm led to the notion that isoform-specific regulators for PP1γ2 in sperm may be responsible for its function. In this report, we studied these inhibitors, PPP1R2, R7, and R11, to determine their spatial and temporal expression in testis and their regulatory functions in sperm. We show that, similar to PP1γ2, the three inhibitors are expressed at high levels in developing spermatogenic cells. However, the transcripts for the regulators are expressed as unique sizes in testis compared with somatic tissues. The three regulators share localization with PP1γ2 in the head and the principal piece of sperm. We show that the association of inhibitors to PP1γ2 changes during epididymal sperm maturation. In immotile caput epididymal sperm, PPP1R2 and PPP1R7 are not bound to PP1γ2, whereas in motile caudal sperm, all three inhibitors are bound as heterodimers or heterotrimers. In caudal sperm from male mice lacking sAC and glycogen synthase kinase 3, where motility and fertility are impaired, the association of PP1γ2 to the inhibitors resembles immature caput sperm. Changes in the association of the regulators with PP1γ2, due to their phosphorylation, are part of biochemical mechanisms responsible for the development of motility and fertilizing ability of sperm during their passage through the epididymis.

The protein phosphatase isoform PP1γ1 substitutes for PP1γ2 to support spermatogenesis but not normal sperm function and fertility†.

Four isoforms of serine/threonine phosphatase type I, PP1α, PP1ß, PP1γ1, and PP1γ2, are derived from three genes. The PP1γ1 and PP1γ2 isoforms are alternately spliced transcripts of the protein phosphatase 1 catalytic subunit gamma gene (Ppp1cc). While PP1γ1 is ubiquitous in somatic cells, PP1γ2 is expressed exclusively in testicular germ cells and sperm. Ppp1cc knockout male mice (-/-), lacking both PP1γ1 and PP1γ2, are sterile due to impaired sperm morphogenesis. Fertility and normal sperm function can be restored by transgenic expression of PP1γ2 alone in testis of Ppp1cc (-/-) mice. The purpose of this study was to determine whether the PP1γ1 isoform is functionally equivalent to PP1γ2 in supporting spermatogenesis and male fertility. Significant levels of transgenic PP1γ1 expression occurred only when the transgene lacked a 1-kb 3΄UTR region immediately following the stop codon of the PP1γ1 transcript. PP1γ1 was also incorporated into sperm at levels comparable to PP1γ2 in sperm from wild-type mice. Spermatogenesis was restored in mice expressing PP1γ1 in the absence of PP1γ2. However, males from the transgenic rescue lines were subfertile. Sperm from the PP1γ1 rescue mice were unable to fertilize eggs in vitro. Intrasperm localization of PP1γ1 and the association of the protein regulators of the phosphatase were altered in epididymal sperm in transgenic PP1γ1 compared to PP1γ2. Thus, the ubiquitous isoform PP1γ1, not normally expressed in differentiating germ cells, could replace PP1γ2 to support spermatogenesis and spermiation. However, PP1γ2, which is the PP1 isoform in mammalian sperm, has an isoform-specific role in supporting normal sperm function and fertility.

Selective ablation of Ppp1cc gene in testicular germ cells causes oligo-teratozoospermia and infertility in mice.

The four isoforms of serine/threonine phosphoprotein phosphatase 1 (PP1), derived from three genes, are among the most conserved proteins known. The Ppp1cc gene encodes two alternatively spliced variants, PP1 gamma1 (PPP1CC1) and PP1 gamma2 (PPP1CC2). Global deletion of the Ppp1cc gene, which causes loss of both isoforms, results in male infertility due to impaired spermatogenesis. This phenotype was assumed to be due to the loss of PPP1CC2, which is abundant in testis. While PPP1CC2 is predominant, other PP1 isoforms are also expressed in testis. Given the significant homology between the four PP1 isoforms, the lack of compensation by the other PP1 isoforms for loss of one, only in testis, is surprising. Here we document, for the first time, expression patterns of the PP1 isoforms in postnatal developing and adult mouse testis. The timing and sites of testis expression of PPP1CC1 and PPP1CC2 in testis are nonoverlapping. PPP1CC2 is the only one of the four PP1 isoforms not detected in sertoli cells and spermatogonia. Conversely, PPP1CC2 may be the only PP1 isoform expressed in postmeiotic germ cells. Deletion of the Ppp1cc gene in germ cells at the differentiated spermatogonia stage of development and beyond in Stra8 promoter-driven Cre transgenic mice results in oligo-terato-asthenozoospermia and male infertility, thus phenocopying global Ppp1cc null (-/-) mice. Taken together, these results confirm that spermatogenic defects observed in the global Ppp1cc knockout mice and in mice expressing low levels of PPP1CC2 in testis are due to compromised functions of PPP1CC2 in meiotic and postmeiotic germ cells.

Isolation, Cryopreservation, and Transplantation of Spermatogonial Stem Cells.

Spermatogonial stem cell (SSC) culture and transplantation pave the way for clinical restoration of fertility in male prepubertal cancer survivors. In this chapter we detail the steps for isolating and freezing testicular tissue along with protocols for the subsequent recovery from cryopreservation and transplantation of cells into a recipient testis. Transplantation of cultured or thawed SSCs provides not only a functional assay for identification of stem cells, a critical tool for the study of the germline stem cell niche in model organisms, but also a framework for reconstitution of spermatogenesis in humans. As proof of concept, the outlined methods have been performed successfully in the murine model and have the potential to be translated to clinical environments.

Significant expression levels of transgenic PPP1CC2 in testis and sperm are required to overcome the male infertility phenotype of Ppp1cc null mice.

PPP1CC2, one of four isoforms of the ser/thr protein phosphatase PP1, is a mammalian-specific splice variant of the Ppp1cc gene, and the only isoform whose expression is confined almost completely to spermatogenic cells. Additionally, PPP1CC2 is the sole isoform found in mammalian spermatozoa. Although PPP1CC1, the other Ppp1cc product, is expressed in many tissues including testis, the only phenotype resulting from deletion of Ppp1cc gene is male infertility. To determine which of the products of Ppp1cc is essential for male fertility, we created two PPP1CC2 transgenes, eTg-G2 and pTg-G2, where Ppp1cc2 expression was driven by the putative endogenous promoter of Ppp1cc or by the testis specific human Pgk2 promoter, respectively. Our results demonstrate that the 2.6-kb genomic region directly upstream of the Ppp1cc structural gene can drive expression of Ppp1cc2, and recapitulate the wild-type tissue specificity of PPP1CC2 in transgenic mice. More importantly, we show that expression of PPP1CC2 alone, via either promoter, is able not only to restore normal spermatogenesis, but the fertility of Ppp1cc null mice as well, provided that transgenic PPP1CC2 expression in testis reaches at least a lower threshold level equivalent to approximately 50% of its expression by a Ppp1cc +/- male. We conclude that the endogenous Ppp1cc promoter normally functions in the testis to maintain a sufficient level of PPP1CC2 expression for normal spermatogenesis to occur, and that production of spermatozoa capable of fertilization in vivo can take place in the complete absence of PPP1CC1 expression.

Observations on the extragenital effects of oestrogen in females: a report.

Some extragenital effects of the female sex steroids are known for a long time and serious research is going on in this matter. A study was carried out at Medical College, Kolkata on 10 female patients suffering from perimenopausal syndrome--they were administered synthetic oestrogen tablets and the oestrogen-induced changes were noted on some parameters ie, psycho-analytical score, electroencephalograph, lung function tests, electrocardiograph, blood female sex hormone level. On the basis of the study it was observed that with low doses of oestrogen (ethinyloestradiol 0.5 mg once daily) the changes were not marked enough. But when it is compared with animal study, where it causes appreciable changes, it can safely be concluded that oestrogen causes (i) some desynchronisation (faster rhythm, lesser voltage) of EEG, (ii) bradycardia/increased sinus arrhythmia and increased amplitude of ventricular complex in ECG, (iii) reduction of scoring rate for anxiety and depression.